MICROBIOLOGY NOTES

BACTERIOLOGY

BACTERIA
A. Prokaryotic organisms
1. Bacteria are ________ organisms that lack a true nucleus and nuclear membrane. The lack of mitochondria, endoplasmic
reticulum, and Golgi bodies also differentiates the prokaryotic bacteria from eukaryotes.

2. The bacterial genome is a single, closed, circular chromosome of double-stranded DNA called the nucleoid. Plasmids are small
circular molecules of extrachromosomal circular DNA. Antibiotic resistance is often encoded by genes located on plasmids.
Chromosomal or plasmid gene exchange via transformation, transduction, or conjugation are means of genetic recombination.

3. Reproduction of bacteria is achieved by binary fission, a means of asexual reproduction. Prokaryotes do not reproduce sexually.

4. Energy generation of bacteria is cytoplasmic membrane-associated via the electron transport chain.
5. Bacteria usually range in size from 0.2 to 2 um in diameter and 1 to 6 um in length. The four basic morphological types are cocci
(spherical-shaped cells), bacilli (rod-shaped cells), spirilla (spiral-shaped cells), and vibrios (comma-shaped cells (Figure 7–1).

B. Growth and nutrition

1. Prokaryotic bacteria have three major nutritional needs for growth.
a. A source of carbon is needed for the synthesis of cellular constituents.
b. A source of nitrogen is necessary for the synthesis of protein.
c. Energy (ATP) is needed for cellular functions.
2. The optimum pH for the growth of most bacteria is 7.0 to 7.5. Acidophiles grow at an acidic pH and alkaliphiles grow optimally
at an alkaline pH.

3. A wide range of growth temperatures facilitate the growth of bacteria. The optimum Growth temperature for most human
bacterial pathogens is 35◦C to 37◦C, the temperature of the human body.

4. Bacteria may be classified based on the atmospheric requirement for the growth.
a. Obligate aerobes require oxygen for growth.
b. Aerotolerant aerobes can grow in the presence of oxygen, but grow best in an anaerobic environment.
c. Facultative anaerobes grow in both aerobic and anaerobic environments.
d. Obligate anaerobes cannot grow in the presence of oxygen.
e. Capnophilic bacteria require concentrations of 5% to 10% CO2 for optimal growth.
f. Microaerophilic bacteria grow optimally in a reduced level of oxyge

BIOSAFETY LEVELS
LEVEL RISK TYPES OF AGENTS EXAMPLES OF AGENTS PRECAUTIONS
Bacillus subtilis,
Mycobacterium Standard microbiological
Minimal Those not known to cause
I gordonae practices. No special
disease in healthy adults
(AKA_____________), soil equipment
microbes
BSC I or II and PPE
Moderate Common human _______, _______, _____, Autoclave must be available.
II
pathogens HBV, influenza Limited
access. ____________
______________, Francisella,
Those that may cause Brucella,
Same as above plus negative
High serious or lethal disease Mycobacterium tuberculosis,
III air flow,
via inhalation. Rickettsia
sealed windows.
_____________________ rickettsii, Coxiella burnetii,
mold stages of systemic fungi
Requires use of class III BSC;
full-body, air-supplied positive
Those that pose high risk of pressure suit; independent
Ebola virus, Lassa virus,
life-threatening disease. May unit with specialized
IV ____________ others that cause
be transmitted by aerosols. ventilation & waste
hemorrhagic fevers
______________________ management to
prevent release into
environment.

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BIOLOGICAL SAFETY CABINETS
CLASSIFICATION DESCRIPTION USE

Open front. Unsterilized room air enters. Air passes

Provides minimal personnel protection.
I through HEPA or _______________________ filter before
Doesn’t protect work surface.
being exhausted.

Laminar flow cabinets with variable sash opening. Air Type most commonly used in hospital
II passes through 1 HEPA filter before reaching work surface micro labs. Provides protection for
& 2nd one before being exhausted. worker & work

Completely enclosed. Negative pressure. Air is filter Provides maximum protection. Used in
III sterilized coming in & going out. Gloves are attached to labs that work with extremely
front. hazardous organisms.

STERILIZATION AND DISINFECTION

A. TEMPERATURE - Most reliable method
 Steam under Pressure - Ex: _________. Most practical & dependable method. 15 lb of
pressure for 15 min. 121°C. Kills spores
 Boiling - Spores may not be killed
 Pasteurization - Used in food industry to kill foodborne pathogens. Doesn’t
sterilize. ___________________________
 Inceniration
- Used to sterilize __________________________

B. FILTRATION - Used to sterilize liquids that are thermolabile (e.g., ______).

HEPA air filters are used in BSC
C. RADIATION - UV light. Used in BSC
(How many hours? _________________)
D. CHEMICALS
 Alcohol - Ethyl & isopropyl (70%–80%) frequently used as antiseptics &
disinfectants. Don’t kill spores
 Chlorine
- Sodium hypochlorite (household bleach). One of most effective
agents against HIV & HBV. 10% solution. Prepare daily

 H2O2 - 3% solution used as antiseptic

 Iodine & iodophors
- Iodophors (e.g., povidone-iodine) release iodine slowly & are
less irritating & non staining. Effective skin antiseptics

 Quaternary ammonium
- _________________________. Not effective against spores,
compounds
mycobacteria, or non-enveloped viruses. Used to disinfect
floors, walls, furniture

E. GAS
 Ethylene glycol Widely used in hospitals to sterilize materials that can’t withstand
steam

BACTERIAL TOXINS
EXOTOXIN ENDOTOXIN
Produced by: Gram (+) Gram (-)
Location in cell: within or without within
Composition: Lipopolysaccharide
Protein (Specifically:__________)
(Specifically: _________)

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Effect: ___________ ___________
Toxicity: High Low
Stability to Heating: (that’s why cook
___________ ___________
canned goods)
Antigenicity: High Low
Converted to Toxoid? Yes No

SPECIMEN COLLECTION GUIDELINES

 Obtain during acute phase of infection (within 2–3 days for viruses).
 Collect ______antibiotics are administered
 Sample appropriate site (in the Lab why sometimes 2 sites?)

 Use swabs with Dacron or polyester tips & plastic shafts. _________________________
(Specifically to what organism?)

 Properly label container, not lid (Why?)

 Deliver to lab within 30 min of collection
 For anaerobes, aspirates preferred to swabs
 For prolonged transit, use special preservatives or holding media
SPECIMEN PRESERVATION AND STORAGE
SPECIMEN STORAGE TEMP. (UNPRESERVED) PRESERVATIVE OR TRANSPORT MEDIA
Sputum 4°C None
Transport media, e.g., Cary-Blair transport
4°C. Best to process without delay. Refrigeration
Stool for culture medium if specimen can’t be cultured within
may kill Shigella
2 hr of collection
Depending on procedures to be performed:
polyvinyl alcohol (PVA), 10% formalin, sodium
Can hold formed specimens at 4°C. Sof
Stool for ova & acetate–acetic acid–formalin (SAF),
or liquid specimens should be examined
parasites merthiolate-iodine-formalin (MIF), others
immediately or preserved
without formaldehyde or mercury (e.g.,
Ecofix, Parasafe)
Transport tubes with boric acid–glycerol if
Urine specimen can’t be processed within 2 hr of 4°C for not more than 24 hr
collection
CSF 25°C or 35°C. Don’t refrigerate (Why?_______) None
Specimen w/
25°C. Don’t refrigerate Anaerobic transport systems to eliminate O2
Anaerobes
Amies transport medium with charcoal,
Neisseria gonorrhoeae Transgrow, JEMBEC plates, Gono-Pak, BioBag 25°C. Don’t refrigerate (why?)
(best to inoculate medium directly)
Viruses Viral transport medium 4°C. For delay >24 hr, freeze at −70°C

GRAM STAINING
REAGENT FUNCTION OTHER
Crystal Violet Basic dye/ Primary stain Stains all bacteria purple
Iodine Mordant _______________________
Alcohol (95% EA or Acetone) Decolorizer Removes crystal violet from Gram (-)
Safranin Counter stain/ Secondary stain Stains Gram (-) pink
What is the most crucial step?

STAINING PROPERTIES OF GRAM (+) AND GRAM (-) BACTERIA

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 GRAM REACTION CELL WALL STAINED BY COLOR IN GRAM STAIN
Thick peptidoglycan layer with teichoic
acid & lipoteichoic acid. Teichoic acid
Gram (+) Crystal violet Purple
cross-links prevent decolorization in
Gram stain.

Thin peptidoglycan layer covered with

proteins, phospholipids, &
lipopolysaccharides. Decolorizer
Gram (-) ____________ Pink
causes ↑ permeability of lipid-rich cell
wall. Primary stain (crystal violet)
washes out.

STAINS
A. Gram’s stain
1. Crystal violet is the primary stain. Iodine binds the crystal violet to the cell wall (mordant). Decolorizer washes out any unbound
dye. Safranin O is a counterstain.
a. Gram-positive cells retain the crystal violet and stain purple
b. Gram-negative cells are decolorized, retain the safranin O counterstain, and stain red or pink

2. Clinical use. Gram’s stain is especially useful for examining smears of clinical specimens. Initial treatment, and often a
presumptive identification, can be made from Gram’s stain results. White and red blood cells (WBCs and RBCs), as well as
cellular debris, stain pink. This can serve as an internal control.

B. Acridine orange stain is an orange fluorescent stain used to detect bacteria in body fluids in which numbers of bacteria may be few (e.g.,
blood and spinal fluid). The stain can also be used to detect bacteria in direct smears with excess cellular debris. It is very sensitive
and can detect small numbers of bacteria that are living or dead. The procedure consists of flooding a methanol-fixed smear with acridine
orange for 2 minutes, washing, and then observing with a fluorescence microscope

C. Methylene blue stain is especially helpful for demonstrating metachromatic granules and characteristic morphology of Corynebacterium
diphtheriae from Loeffler coagulated serum medium. The procedure consists of flooding a fixed smear with methylene blue, followed
by washing

D. Acid-fast stain is used to detect organisms that do not stain well with other conventional stains (e.g., Mycobacterium spp., Nocardia,
Actinomyces). These organisms have a high lipid content in their cell walls. Once stained, they are very resistant to decolorization by
acid alcohol. The most commonly used method is a carbolfuchsin stain, the modified Kinyoun stain. The primary stain is carbolfuchsin,
which contains a surface-active detergent to facilitate penetration of the stain without heating. After washing, methylene blue is used as a
counterstain. Acid-fast organisms appear red against a blue background. The Ziehl-Neelsen is a carbolfuchsin stain that utilizes heat to drive
the stain into the mycobacterial cell wall

E. Auramine-rhodamine stain is a fluorescent stain that detects mycolic acids and can be used for staining acid-fast organisms. The smear is
stained with auramine-rhodamine, decolorized with acid alcohol, and then flooded with potassium permanganate. It is observed with a
fluorescence microscope. The cells appear yellow against a dark background

F. Calcofluor white stain binds specifically to chitin, which is found in fungal cell walls. It is used to detect yeast cells and hyphae in skin
scrapings and other specimens. The fungal elements appear green or blue-white

TYPES OF MEDIA (Note! Media may be of more than 1 type)

TYPE EXPLANATION EXAMPLES
Supports growth of
Supportive Nutrient agar, trypticase soy agar
_________________________
Sheep blood agar, chocolate agar, brain-
Contains added growth factors, e.g.,
Enrichment heart infusion, buffered charcoal-yeast
blood, vitamins, yeast extract
extract agar
Columbia colistin–nalidixic acid (CNA)
Contains additives such as dyes,
agar _______________ (EMB),
_______________________ to inhibit
Selective MacConkey, Hektoen enteric (HE),
growth of certain bacteria (e.g.,
xylose lysine deoxycholate (XLD),
gram pos)
Thayer-Martin
Differential Formulated to provide distinct colonial EMB, MacConkey, HE, XLD
appearances based on certain
biochemical rxn (e.g., lactose

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 fermentation, hydrogen sulfide [H2S]
production)

ROUTINE MEDIA FOR AEROBES AND FACULTATIVE ANAEROBES

TYP
MEDIUM
E
Sheep Blood Agar
Chocolate agar (CHOC)
Columbia colistin–nalidixic
acid agar (CNA)
Phenylethyl alcohol agar
(PEA)
Group A–selective strep agar
with 5% sheep blood (SSA)
Eosin methylene blue (EMB)
MacConkey (MAC) agar
Sorbitol MacConkey
(SMAC) agar
Hektoen enteric (HE) agar
Xylose lysine deoxycholate
(XLD)
Salmonella-Shigella (SS) agar
Gram-negative broth (GN)
Deoxycholate-citrate agar
Campylobacter broth
Campylobacter blood agar
(Campy BAP)
ISOLATION FOR
E, D Most nonfastidious bacteria
E Haemophilus & Neisseria
S GP
S GPC & anaerobic GNR
Group A strep from respiratory
S
sources
S, D Enteric GNR
S, D Enteric GNR
S E. coli O157:H7
S, D Salmonella & Shigella in stool
S, D Salmonella & Shigella in stooL
S Salmonella & Shigella in stool
Salmonella & Shigella from
E, S
stools & rectal swabs
S Salmonella & Shigella
E Campylobacter from stool
E,S Campylobacter from stool
OTHERS
Tryptic soy agar with 5% sheep blood. Allows
differentiation of hemolysis
Supplies X & V factors. Incubate in ↑ CO (What
organism requires X and V factor? _____)
Colistin & nalidixic acid suppress most GN.
Contain 5% sheep blood
Phenylethyl alcohol inhibits enteric GNR. Contains
5% sheep blood.
Contains antibiotics to suppress normal throat
flora. Group B strep will also grow
Eosin & methylene blue inhibit GP. Lactose
fermenters (LF) green-black or purple. E. coli
produces green metallic sheen. Non–lactose
fermenters (NLF) colorless.
Bile salts & crystal violet inhibit most GP. LF pink.
NLF colorless
E. coli O157:H7 doesn’t ferment sorbitol.
Colorless colonies. Some labs have stopped using
because non-O157 serotypes can be pathogens
Bile salts, bromothymol blue, & acid fuchsin
inhibit normal GI flora. Nonpathogens orange to
salmon pink. NLF green to blue-green. H2S-pos
colonies have black precipitate.
Deoxycholate inhibits many GNR & GP. 4 types of
colonies: yellow (e.g., E. coli), yellow with black
centers (e.g., some Proteus species), colorless or
red colonies (e.g., Shigella), red colonies with
black centers (e.g., Salmonella). (Some shigellae
may be inhibited. Some salmonellae may not
produce H2S.)
Brilliant green & bile salts inhibit other
enterics. Salmonella & Shigella don’t ferment
lactose (colorless colonies). Salmonella produces
H2S (black center)
Deoxycholate & citrate salts retard growth of GP.
Subculture onto selective differential agar after 6–
8 hr & 18–24 hr incubation. Use of enrichment
broths for stool cultures is decreasing
Other enterics inhibited
Subculture to Campy-selective agar after
overnight incubation at 4°C
Incubate plates in ↑ CO2 at 42°C

Selective Media for Isolation of Neisseria gonorrhoeae and Neisseria meningitidis

MEDIUM COMMENTS
Vancomycin, colistin, nystatin, & trimethoprim inhibit growth of other bacteria & fungi.
Modified Thayer-Martin (TM)
Incubate in ↑ CO 2. Some N. gonorrhoeae may be inhibited
Martin-Lewis Similar to Thayer-Martin, but different antibiotics. Inhibits yeast better. Incubate in ↑ CO2.
Incubate in ↑ CO
New York City medium (NYC)
2. Some N. gonorrhoeae are inhibited by antibiotics. Genital mycoplasmas will grow.
GC-LECT Antibiotics to inhibit GN & GP bacteria & yeast.
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 For transportation & growth of N. gonorrhoeae. Plates contain Neisseria-selective medium
JEMBEC plates & come with resealable polyethylene bag & CO2-generating tablet. No need to transfer to
culture plate.

SPECIAL BACTERIOLOGIC MEDIA

MEDIUM
Cystine-tellurite blood agar
Loeffler medium
Tindale agar
Bismuth sulfite agar
Cefsulodin-irgasan-
novobiocin (CIN) agar
Alkaline peptone water
(APW)
Thiosulfate citrate bile salts
sucrose (TCBS) agar
Bordet-Gengou agar
Regan-Lowe agar
Buffered charcoal-yeast
extract (BCYE) agar
Human blood bilayer Tween
(HBT) agar
USE
Differential medium for isolation of
Corynebacterium diphtheriae
Enrichment medium for C. diphtheriae
Selective differential medium for C.
diphtheriae
Selective for Salmonella
Selective medium for Yersinia
inhibits GPC. Cefsulodin inhibits most GP & GN. Y.
enterocolitica, Aeromonas, &
Plesiomonas shigelloides
“bull’s-eye” colonies surrounded by colorless halo.
Enrichment medium for recovery of
Vibrio from stool
Selective for Vibrio
Selective enrichment medium for
isolation of Bordetella pertussis
Selective for B. pertussis
Enrichment medium for isolation of
Legionella
Selective & differential for G. vaginalis
OTHERS
C. diphtheriae produces black colonies
Promotes development of metachromatic
granules
Corynebacterium spp produce gray to black
colonies due to reduction of tellurite. C.
diphtheriae colonies are surrounded by a brown
halo
Bismuth sulfite & brilliant green inhibit most
others. S. typhi colonies are black, surrounded by
metallic sheen. Others are light green. (Some
salmonellae may be inhibited.)
Crystal violet inhibits most GN. Novobiocin
enterocolitica ferments mannitol, appears as red
Alkaline pH suppresses commensals. Subcultured
to TCBS
High pH inhibits most bacteria. V. cholerae
ferments sucrose, produces yellow colonies. V.
parahaemolyticus & V. vulnificus don’t ferment
sucrose; usually produce blue-green colonies
Potato-glycerol-based medium enriched with
blood. Contaminants inhibited by methicillin.
“Cough plate.” Bordetella colonies resemble
mercury droplets.
Charcoal agar supplemented with horse blood,
cephalexin, & amphotericin B
Yeast extract & L-cysteine enhance growth of
Legionella. Charcoal absorbs toxic compounds.
Incubate in ↑ CO 2 for 48 hr. Colonies are beta
hemolytic.

MICROCOCCACEAE
A. General characteristics
1. Members of the family Micrococcaceae are gram-positive cocci, aerobic or facultative anaerobes, and catalase-positive (except
Stomatococcus).
a. The catalase test differentiates the Micrococcaceae from the gram-positive, catalase-negative Streptococcaceae.
b. Hydrogen peroxide is converted to water and oxygen in the presence of the bacterial enzyme catalase. The observation
of vigorous bubbling when the bacterium is mixed with a drop of 3% hydrogen peroxide is a positive test
2. Most are members of the indigenous flora and are commonly isolated from a wide variety of diseases.
3. Staphylococcus
a. The staphylococci are catalase-positive, nonmotile, facultative anaerobes that are normal inhabitants of the skin and
mucous membranes. These organisms commonly cause human infections.
b. Species are initially differentiated by the coagulase test, the most important coagulase-positive species is S. aureus.
Some animal species produce coagulase, but are rarely isolated from human samples.
c. Staphylococci that do not produce coagulase are called “coagulase-negative staphylococci.” The most prominent
species are S. epidermidis and S. saprophyticus.
4. Micrococcus are opportunistic pathogens found only in immunocompromised persons with low pathogenic significance, but may
be isolated as a contaminant or as part of the normal flora.
5. Stomatococcus (Table 7–1; Figure 7–4). This genus is part of the normal oral flora and is rarely isolated from infection. The
colonies adhere strongly to the agar surface.

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STAPHYLOCOCCI
ORGANISM PATHOGENECITY GRAM STAIN SBA COLONIES KEY CHARACTERISTICS OTHERS
Catalase (+)
Normal flora of skin,
1–3 mm, round, Fermentative.
mouth, pharynx,
GPC, usually smooth, convex, Microdase neg
In General, vagina, urethra, GI
See specific in CLUSTERS glistening, (modified oxidase)
Staphylococcus tract. Facultative
organism below except opaque, entire Resistant to Bacitracin.
spp anaerobe. Grows on
Micrococcus edge, butyrous Susceptible to
most nonselective
(butter-like). furazolidone &
media. Salt tolerant
lysostaphin
S. aureus Causes Same Most are Coagulase (+) 10%–60% are
suppurative _____________ ________________ carriers.
cutaneous (small zone). (yellow colonies on Spread by direct
infections, toxic May be golden. mannitol salt agar). contact.
shock syndrome, Usually DNase &
food thermonuclease (+),
poisoning 85%–90% resistant to
penicillin.
Coagulase- Opportunistic Same White, usually Coagulase (-), Grow Normal on skin &
negative pathogen. ______________ on mannitol salt agar Mucous membranes.
staphylococci Common but don’t ferment Often contaminant.
(CNS) cause of mannitol. Sensitive to Usually only
hospitalacquired novobiocin speciated if from
UTI normally sterile
site. 50%–80% are
S. epidermidis.
S. saprophyticus UTI in young Same White to slightly Coagulase (-) Novobiocin only
sexually active yellow. Non ________________ performed when CNS
females, hemolytic. May ferment mannitol. isolated from urine
urethritis & of
prostatitis in female
males
Micrococcus Usually Large GPC in Often pigmented Catalase (+) Not commonly
nonpathogenic. pairs, tetrads (bright yellow, Coagulase (-) isolated. Must
Found in (predominant orange, pink, Oxidative Microdase differentiate from
environment & arrangement), tan). High- (+) (modified oxidase) staph. Usually only
on skin, mucous clusters domed colonies Susceptible ________. grow aerobically
membranes, Resistant to
oropharynx __________________

TESTS FOR STAPHYLOCOCCI

TEST PRINCIPLE KEY REACTIONS OTHERS
Catalase Enzyme catalase converts 3% Staphylococci pos. Bubbles after 20–30 sec
hydrogen peroxide (H2O2) to Streptococci & enterococci aren’t considered pos rxn.
oxygen & water. Immediate neg Catalase from RBCs in blood
bubbling agar may produce weak
bubbles.
Coagulase Enzyme coagulase causes S. aureus pos Slide test is screening test.

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(DIFFFERENTIATE:
______________________)
Slide agglutination tests
for S. aureus
Mannitol salt agar (MSA)
Novobiocin susceptibility
coagulation (tube test) or
agglutination (slide test) in
plasma
Agglutination of latex beads
coated with fibrinogen & abs
to protein A (protein in cell
wall of S. aureus)
Fermentation of mannitol
results in color change from
pink to yellow.
Organisms resistant to
novobiocin grow to edge of
disk
S. aureus pos
S. aureus pos
S. saprophyticus is resistant.
Other CNS susceptible
Detects bound coagulase
(clumping factor). If neg,
tube test should be
performed. Detects free
coagulase. Largely replaced
by latex agglutination tests
Most labs report pos
organisms as S. aureus
7.5% salt inhibits most
organisms other than staph.
All staph can grow on MSA.
Rarely used for ID of S.
aureus today because other
species ferment mannitol.
Performed on CNS isolated
from urine

STREPTOCOCCUS, ENTEROCOCCUS AND RELATED GENERA

A. General characteristics
1. This group are catalase-negative, gram-positive cocci (old cells may stain gram-negative or gram-variable) that are
arranged in pairs or chains and are facultative anaerobes. Growth requirements may be complex, and the use of blood
or enriched medium is necessary for isolation. Their role in human disease ranges from well-established and common,
to rare but increasing.
2. Hemolysis patterns on sheep blood agar
HEMOLYSIS DESCRIPTION EXPLANATION EXAMPLES
Streptococcus pneumonia,
Green zone around colony.
Alpha __________________ Viridans streptococci, some
May be narrow or wide.
enterococci
Clear zone around colony. Group A strep, group B strep,
Beta Complete lysis of RBCs
May be narrow or wide. Listeria monocytogenes
Gamma (non- hemolytic) No zone of hemolysis ________________ Some enterococci

STREPTOCOCCI
KEY
ORGANISM PATHOGENECITY GRAM STAIN COLONIES ON SBA OTHERS
CHARACTERISTICS
In General, See specific Oval GPC in <1 mm, white to Catalase neg Facultative

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 anaerobes.
gray, translucent,
Streptococcus chains Require enriched
organism below or semiopaque.
spp & pairs media. Chaining
Variable emolysis
besting broth cultures
Most common is
Causes 90% of S. pyogenes.
strep infections. hemolysis due to O2-
Strep sore throat, stable streptolysin S
rheumatic fever, Pinpoint. Grayish & O2- labile
Group A glomerulonephritis white. Translucent. Sensitive to streptolysin O.
Oval GPC in
streptococci , Usually beta _________ To detect species that
chains
(GAS) scarlet fever hemolytic (wide Resistant to ______ produce streptolysin
(scarlatina), zone). O only, stab into
erysipelas, agar, place coverslip
puerperal sepsis, over inoculum, or
impetigo incubate
anaerobically
S. agalactiae. Vaginal
& rectal swabs
Normal flora of collected
Slightly larger than
female genital from pregnant
GAS. Gray-white. Resistant to SXT &
tract. Most women at 35–37 wk
Narrow zone of bacitracin.
___________ common cause of Same gestation.
diffuse beta Sodium hippurate
neonatal Inoculated in
hemolysis. May pos. CAMP pos
septicemia & selective broth, e.g.,
be nonhemolytic.
meningitis LIM, GBS
broth, StrepB Carrot
Broth
Normal in GI tract.
Usually Most common is
Group D Causes nosocomial
nonhemolytic. S. gallolyticus
streptococci, UTI, wound Same Hydrolyzes esculin
May be alpha (formerly
nonenterococci infections,
hemolytic S. bovis).
bacteremia
Normal in mouth,
GI tract, female
genital tract. Oval GPC in Usually alpha or Hydrolyzes esculin. Lancefield group D.
Enterococcus Causes nosocomial pairs & nonhemolytic. Grows in 6.5% NaCl 80% are E. faecalis,
UTI, wound chains Rarely beta broth. PYR pos 15% E. faecium
infections,
bacteremia
Normal in upper
Football- Round,
respiratory tract
shaped translucent,
of some. Most
(lancet- glistening, dome
common cause
shaped) shaped when
of community
GPC. Usually young. Central Bile solubility _____ No Lancefield group.
Streptococcus acquired
in pairs. May depression with Sensitive to >80 serotypes based
pneumoniae pneumonia. Major
be single or age (umbilicate) ________ on capsular antigens.
cause of otitis
in short due to autolysis.
media,
chains. Alpha hemolytic.
meningitis in
Frequently Encapsulated
adults. Infects
encapsulated strains are mucoid
sinuses, eyes
Viridans Normal in oral, GPC in Usually alpha Resistant to none
streptococci respiratory, GI chains hemolytic, but optochin.
mucosa. may be beta Bile solubility neg
Opportunistic hemolytic or
pathogens. nonhemolytic
Frequent cause of
subacute bacterial
endocarditis.
Gingivitis & dental
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 caries (cavities)

TESTS FOR STREPTOCOCCI

For Beta- Hemolytic Strep
TEST REACTIONS KEY PRINCIPLE OTHERS
Also known as A disk. Performed on
Zone of inhibition after overnight SBA. No longer recommended because
GAS susceptible. GBS
____________ incubation = susceptibility to some groups C & G are susceptible.
resistant
bacitracin Replaced by PYR & serogrouping by latex
agglutination.
Trimethoprim- GAS & GBS resistant. Used in conjunction with bacitracin
Organisms resistant to SXT grow
sulfamethoxazole Group C & G strep disk to differentiate GAS from group C or
up to disk
(SXT) disk susceptible G. Requires overnight incubation.
More specific than bacitracin for GAS.
Pyrrolidonase If PYR is hydrolyzed, red color GAS pos (also
GAS is only beta-hemolytic strep that’s
(PYR) test after addition of color developer enterococci)
PYR pos. Disk test only takes minutes
Classic method: unknown streaked
perpendicular to streak of b -lysin
producing S. aureus. Incubated in
ambient air overnight. False pos in CO2.
GBS produces extracellular protein Arrowhead hemolysis where inoculum
____________ that enhances hemolysis of GBS pos lines meet. Disks Containing b -lysin can
betahemolytic S. aureus on SBA. be used instead of S. aureus.

Rapid test:
drop of b -lysin on colonies on SBA.
Only requires 20-min incubation.
Organisms that produce
Hippurate hippuricase (hippurate hydrolase)
GBS pos Alternative to CAMP. 2-hr test available
hydrolysis hydrolyze sodium hippurate to
benzoate & glycine.
Latex particles coated with Organisms Tests commercially available for rapid ID
Slide
groupspecific antibodies agglutinate in of groups A, B, C, D, F, G strep & S.
agglutination
agglutinate in corresponding pneumoniae. (Most labs only use for ID of
tests
presence of bacterial antigens antisera beta hemolytic.)
FOR ALPHA- HEMOLYTIC STREP
Also known as P disk. Contains
Zone of inhibition ‡ 14 mm (using S. pneumoniae ethylhydrocupreine hydrochloride. Placed
6-mm disk) OR ‡ 16 mm (using susceptible. on lawn of inoculum on SBA.
Optochin disk
10-mm disk) = susceptibility to Viridans strep Plates incubated overnight in ↑ CO2. If
Optochin resistant zone of inhibition <14 mm, ID organism as
S. pneumonia only if bile soluble
Can be performed in broth or on colony.
Bile salts (e.g., sodium
Positive = clearing of broth or
Bile solubility deoxycholate) cause lysis of some S. pneumoniae pos
disappearance of colony. Results in 30
Organisms
min or less
FOR NON- HEMOLYTIC STREP/ ENTEROCCOCI
Pyrrolidonase See “Tests for Identification of Enterococci pos (also
none
(PYR) test Beta-Hemolytic Streptococci” GAS)
Organisms that can grow in
40% bile & produce esculinase Group D strep & Bile inhibits GPs other than group D
____________
hydrolyse esculin, producing enterococci pos strep & enterococci
black precipitate
Organisms that produce esculinase
Group D strep &
Esculin test hydrolyze esculin on disk, Doesn’t test for bile tolerance.
enterococci pos
producing dark spot
Enterococci pos (also
Organisms that can grow in
___________ GBS). Group D strep Requires overnight incubation.
6.5% NaCl produce turbidity
neg

10 | m a d e b y M C P M , R M T
STREPTOCOCCUS- LIKE ORGANISMS

1. Aerococcus is very similar to Enterococcus on blood agar. The gram-positive coccus is susceptible to vancomycin and can be
isolated from tissue samples of endocarditis and other varied infections.
2. Leuconostoc is very similar to viridans streptococci on blood agar. It is found in the general environment. A Gram’s stain
shows gram-positive coccobacilli in pairs and short chains. Leuconostoc has been found in patients who have
meningitis and endocarditis. It is intrinsically resistant to vancomycin.
3. Pediococcus is also found in the general environment. A Gram’s stain shows grampositive cocci in pairs, tetrads, and
clusters. Pediococcus is a rare isolate in patients who have septicemia. The bacterium is intrinsically resistant to
vancomycin

LAP test (leucine aminopeptidase) is used to help differentiate Aerococcus and Leuconostoc from the other Streptococcus
species. Both bacteria are LAP-negative, while other streptococci are LAP-positive. LAP hydrolyzes the substrate, leucine-
Bnaphthylamide, to B-naphthylamine. Development of a red color is detected upon
addition of DMACA

AEROBIC GRAM POSITIVE BACILLI

General characteristics
1. The members of this group that are seen most frequently in the clinical laboratory
2. Except for Corynebacterium diphtheriae, these organisms are of low pathogenicity and usually require an
immunocompromised host.
3. With the exception of Bacillus, these organisms are all pleomorphic rods, and most grow well on standard media.

AEROBIC SPORE FORMING GRAM POSITIVE RODS

KEY
ORGANISM PATHOGENICITY GRAM STAIN CULTURE OTHERS
CHARACTERISTICS
Large spreading beta
hemolytic colonies
In General, Usually See B. anthracis with irregular edges Catalase pos. Must rule out
Bacillus spp contaminants below. (Medusa head). Most are motile B. anthracis.
Whitish gray. May
be pigmented

One of most Large with Large, adherent, Nonhemolytic. Potential

highly pathogenic square ends. Non hemolytic, flat Catalase pos. bioterrorism
microorganisms. May be in chains. to slightly convex, Nonmotile. organism.
Causes anthrax. Oval, central to irregular border, ________ seen in Handle in BSC.
Contracted from subterminal ground-glass CSF & blood Submit to public
__________ contaminated spores that appearance, comma- smears health lab for
hides, wool, aren’t swollen. shaped projections. confirmation
meat. Rare in U.S Looks like Stands up like beaten
bamboo. Spores egg white when (What BSC is used?)
may not be seen touched with loop
indirect smear
Can Same as Same as B. anthracis Hemolytic. Motile. 2nd most important
cause local & B. anthracis except hemolytic pathogen in genus
Bacillus cereus systemic
infections, food (seen in fried rice)
poisoning
AEROBIC NON- SPORE FORMING GRAM POSITIVE RODS
KEY
ORGANISM PATHOGENICITY GRAM STAIN CULTURE CHARACTERISTIC OTHERS
S
Corynebacterium Normal on skin & Irregular, Facultative ____________
spp mucous membranes. slightly anaerobes. ____________
11 | m a d e b y M C P M , R M T
 curved,
nonparallel
sides, club-
Opportunistic shaped ends. Grow on most
pathogens Chinese letter media. (VS Bacillus)
or picket fence
formation
(palisades)
Gray-black
colonies with
brown halos on
Tindale agar.
ID requires
Black colonies
demonstration of
on cystine
Catalase pos. toxin production.
Corynebacterium Diphtheria. Rare in tellurite.
Same as above Nonmotile. Modified Elek
diphtheriae U.S. Loeffler
Toxin producing immunoprecipitation
medium
test. PCR for TOX
stimulates
gene
growth &
production of
metachromatic
granules.

Most commonly
Same as other Catalase pos.
Corynebacterium Hospital-acquired isolated diphtheroid.
Same as above commensal Rapid sucrose
jeikeium pathogen. Highly resistant to
corynebacteria urea (RSU) neg
antibiotics
Catalase pos.
Hippurate
hydrolysis pos.
Grows from 0.5°–
Tiny colonies Esculin pos.
Parallel sides, 45°C. Cold
Meningitis & with narrow CAMP pos
rounded ends, Enrichment may
septicemia in zone of (hemolysis
Listeria coccobacillary. be used. Catalase
newborns & indistinct looks like shovel,
monocytogenes Singles, chains, differentiates
immunocompromised. beta hemolysis. not arrowhead).
or diphtheroid from GBS. Motility
Food poisoning Translucent, Tumbling motility
arrangement differentiates from
gray on wet mount.
diphtheroids
Umbrella growth
in motility agar at
RT but not at 35°C

Aerobic Actinomycetes include Nocardia, Actinomadura, and Streptomyces species. These microorganisms are similar
morphologically to fungi. They are gram-positive bacilli, but do not produce spores.

1. Actinomadura are etiologic agents of mycetomas. Their microscopic and macroscopic morphology is similar to that
12 | m a d e b y M C P M , R M T
 of the Nocardia species. However, the microorganisms are not acid-fast.

2. Streptomyces species are primarily saprophytes, found as soil organisms. They may cause opportunistic infections
similar to those caused by the other aerobic actinomycetes.

3. Nocardia see below…

Slow-growing.
N. brasiliensis most
Immunocompetent: On SBA
common species
skin infections. Fine branching wrinkled, Catalase pos.
to cause skin
Immunocompromised: filaments with dry, crumbly, Partially
Nocardia infections. N.
invasive pulmonary fragmentation. chalky white to acid fast. Sulfur
asteroids most
& disseminated Often beaded. orange-tan, granules
Common species to
infections beta
cause lung infections
hemolytic
Erysipelothrix rhusiopathiae is an uncommon isolate. It is a pleomorphic gram-positive bacillus that often forms long
filaments (see Web Color Image 7–30). The usual route of infection is through the skin. It is catalase negative, and forms
hydrogen sulfide. The bacterium is nonmotile, but produces a characteristic “bottle brush” extension laterally from the
streak line in soft gelatin agar
Lactobacillus species is a pleomorphic, nonspore-forming, gram-positive bacillus. It is catalase-negative, and may grow
better under anaerobic conditions. Lactobacilli are normal vaginal flora
Arcanobacterium haemolyticum is a small, β-hemolytic, nonspore-forming, grampositive bacillus. It resembles the β-
hemolytic streptococci. The organism is the etiologic agent of pharyngitis and must be differentiated from Streptocococcus
pyogenes. It is catalase-negative and exhibits a reverse CAMP reaction.

GRAM NEGATIVE COCCI AND DIPLOCOCCI (Neisseria and Moraxella)

General characteristics
1. Members of this group are gram-negative cocci that are often seen in pairs. The adjacent sides are flattened,
producing a kidney-bean shape
2. They are all oxidase-positive and catalase-positive. Differentiation of species is often based on acid production
from carbohydrate utilization.
3. Some of the pathogenic species may have fastidious growth requirements and capnophilic
4. Proper specimen collection is essential for successful isolation. N. gonorrhoeae and N. meningitidis are especially
sensitive to drying, cold, and chemicals (disinfectants and antiseptics). Dacron or rayon swabs are less inhibitory
than calcium alginate and cotton and are preferred for collection. Specimens should be kept at

KEY
ORGANISM PATHOGENICITY GRAM STAIN CULTURE CHARACTERISTIC OTHERS
S
N. gonorrhoeae Gonorrhea, Intracellular & Requires ↑ CO2. Carbohydrate Susceptible to
salpingitis, extracellular Usually doesn’t utilization: drying & cold. Don’t
ophthalmia of GNDC. grow on SBA. glucose only. refrigerate
the newborn Diagnostic in Grows on CHOC Superoxol pos. specimens (why?)
urethral discharge & Neisseria- Can ID with Molecular methods
from symptomatic selective media. monoclonal for genital
males. Culture Colonies are antibodies specimens. Culture
confirmation small, grayish preferred for non-
required for white or tan. 5 genital specimens
females different colony &
types. May look those from
like mixed culture children. Should
confirm by
different method
13 | m a d e b y M C P M , R M T
N. meningitidis Can be normal Intracellular & Grows on SBA, Carbohydrate Don’t refrigerate.
flora in upper extracellular CHOC, & utilization: Handle in BSC.
respiratory tract. GNDC Neisseria glucose & Serogrouping by
Common cause of selective media. maltose. slide agglutination
meningitis in Colonies are ONPG neg Bacterial antigen
young adults bluish gray or tan. test available for
May be mucoid detection but poor
sensitivity Shouldn’t
replace culture.
Vaccine available
N. lactamica Normal in upper GNDC Will grow on Carbohydrate One of commensal
respiratory tract Neisseria- utilization: Neisseria. Easily
of children. Rare selective glucose, maltose, misidentified as
in adults. Rarely media. Resembles & lactose (slow). N. meningitidis.
causes disease N. meningitidis May
but smaller. react with
meningococcal
typing sera.
ONPG differentiates.

Moraxella Normal in upper GNDC Grows on SBA & ONPG pos. DNase & butyrate
catarrhalis respiratory tract. CHOC. Some may Catalase pos. esterase
Causes respiratory grow at RT &/or Oxidase pos. differentiate
infections in on Neisseria- Carbohydrate from Neisseria spp
young, old, & selective utilization:
compromised; media. “Hockey neg for all sugars
otitis media & puck colonies” (asaccharide) &
sinusitis in (colony can be ONPG. DNase
children pushed over agar pos. Butyrate
surface with loop) esterase pos.

GENERAL CHARACTERISTICS OF ENTEROBACTERIACEAE

 Gram stain:
 Growth characteristic:
 Colonies on sheep blood agar:
 Colonies on MacConkey:
 Biochemical tests:
Non- spore forming gram negative rods
Facultative anaerobes
Most are large, dull, gray, non- hemolytic
Lactose fermenters pink
Non–lactose fermenters colorless
___________________________________________________________

BIOCHEMICAL TESTS FOR ID OF ENTEROBACTERIACEAE

TEST PRINCIPLE INTERPRETATION OTHER
Oxidase Tetramethyl-para-phenylenediamine Pos = blue or purple Good test to differentiate
dihydrochloride reacts with Enterobacteriaceae from
cytochrome C to produce blue or nonfermenters. False pos from
purple color. ironcontaining wire. Use
platinum wire or wooden stick.

Nitrate reduction If organism reduces nitrates to
nitrites, red color develops when
sulfanilic acid & N,N-dimethyl-L
naphthylamine added.
Pos = red, or no color after
addition of zinc dust
If no color develops, zinc dust
added. Reduces nitrates.
No color after zinc dust means
nitrates reduced to N
2 or NO2, i.e., pos rxn.

14 | m a d e b y M C P M , R M T

Carbohydrate When carbohydrate fermented,
fermentation acidic end products cause color
change in pH indicator.
_______________ ONPG is changed to orthonitrophenol
by beta-galactosidase.
Organisms that possess H2S
H2S production producing enzymes produce colorless
H2S gas from sulfur-containing
compounds. H2S reacts with iron
salt in medium to form black
ferrous sulfide.
______________ Sugar fermentation produces acid,
changes color of pH indicator
Indole Tryptophanase deaminates
tryptophan. Indole produced.
Kovacs’s reagent (Paradimethyl-
aminobenzaldehyde) forms pink-
colored complex with indole.
Methyl red (MR) Acid products formed when glucose
is metabolized by mixed acid
fermentation pathway. Color
change in pH indicator.
Voges-Proskauer Acetoin is produced from alternate
(VP) pathway for glucose metabolism
15 | m a d e b y M C P M , R M T
With phenol red indicator,
change from red to yellow
Pos = yellow
Black precipitate
Yellow = acid (A).
Pink = alkaline (K).
Pink butt = glucose
not fermented.
Yellow butt = glucose
fermented.
Pink slant = lactose/sucrose
not fermented.
Yellow slant = lactose and
or sucrose fermented.
Black precipitate = H2S
produced.
Bubbles = gas production
Pos = pink
Pos = red color after
addition of methyl red (pH
indicator)
Pos = red color when
alphanaphthol & KOH
added
__________ after zinc dust
indicates presence of residual
nitrates, i.e., neg rxn.
Frequently tested Carbohydrates
are glucose, lactose, sucrose,
mannose, sorbitol, mannitol,
xylose, adonitol, cellobiose,
dulcitol, trehalose.
All Enterobacteriaceae ferment
glucose.
(that’s why ha TSI mayda yellow)
Test for slow lactose
fermentation. Helpful
in differentiating Citrobacter
(pos) from most Salmonella
(neg).
Sulfur-containing compounds =
sodium thiosulfate, cystine,
methionine.
Good test to differentiate
Salmonella (H2S pos) from
Shigella (H2S neg).
Contains 0.1% glucose, 1%
lactose, 1% sucrose, phenol red,
sodium thiosulfate, iron salt.
Leave cap slightly loose. Record
as slant/butt, e.g., K/A. (Some
record NC [no change] for pink
butt.) Kligler’s iron agar (KIA) is
same except no sucrose
Spot indole test commercially
available.Need source of
tryptophan. Use colonies from
SBA or CHOC agar, not
MacConkey.
Pos = blue. E. coli & Proteus
vulgaris are pos
Little acid produced by this
pathway. Organisms that are VP
pos are usually methyl red neg &
vice versa.
Citrate If organism can use citrate as sole Pos = green to blue, or False neg if cap not loose.
source of carbon, pH ↑, pH indicator growth Klebsiella & Enterobacter are
changes color. pos.

Urease Urease breaks down urea. Ammonia
released, pH ↑, pH indicator
changes color
Phenylalanine Phenylalanine deaminase deaminates
deaminase (PD) phenylalanine to phenylpyruvic acid,
which reacts with ferric chloride to
produce green color.
Decarboxylase If organism has enzyme to
reactions decarboxylate amino acid (e.g.,
ornithine, lysine, arginine), pH ↑,
pH indicator changes color.
Pos = yellow to pink
Pos = green color after
addition of ferric chloride
Pos = yellow to purple
Proteus & Morganella are rapid
urease producers.
Proteus, Providencia, Morganella
are pos
Ornithine decarboxylase (ODC)
differentiates Klebsiella (neg) &
Enterobacter (pos).

Motility Motile organisms grow away from
stab line in motility medium.
Pos = movement away
from stab line or hazy
appearance throughout
medium after overnight
incubation
Most Enterobacteriaceae are
pos, ____________________
Differentiate______________
(neg) & Enterobacter (pos)

ANTIGENS OF ENTEROBACTERIACEAE
ANTIGEN ALTERNATE NAME LOCATION CHARACTERISTICS OTHERS
Lipopolysaccharide, heat Used for serological grouping
O antigen Somatic antigen Cell wall
stable of salmonella & Shigella
H antigen Flagellar antigen Flagella Proteins, heat labile Used to serotype Salmonella.
Role in preventing
Polysaccharide, heat labile,
Phagocytosis. ↑ virulence. Vi
K antigen Capsular antigen Capsule may mask O antigen.
antigen is K antigen produced
Removed by heating
by S. typhi

COMMONLY ISOLATED ENTEROBACTERIACEAE

ORGANISM PATHOGENICITY POSITIVE NEGATIVE OTHER
Escherichia coli UTI, septicemia, Lactose, gas, indole, H2S, VP, citrate, Predominant aerobe
neonatal sepsis & MR, motility PD, urease in GI tract. Most
meningitis, diarrhea common cause of UTI.
(some) Green metallic sheen
on EMB.

Presumptive ID:
lactose pos, dry colony
on MacConkey,
oxidase neg,
indole pos.
E. coli O157:H7
doesn’t ferment
sorbitol; colorless
colonies on SMAC.

________________ Dysentery (shigellosis). MR Lactose, gas, H2S, Blood, mucus, polys in

Most communicable VP, citrate, PD, stool. Rarely
of bacterial diarrheas. urease, motility disseminates. Fragile
Found primarily in organism. S.
crowded or dysenteriae most
Substandard severe.
conditions, e.g., day- Shigella sonnei most
care centers, jails, common in U.S.
prisons Serogrouped by O
antigens. Serogroups

16 | m a d e b y M C P M , R M T
 A, B, C, D. Closely
related to Escherichia
on molecular basis
Edwardsiella tarda Opportunistic. Gas, H2S, indole, MR, Lactose, VP, Chief reservoirs are
Bacteremia, motility citrate, PD, urease reptiles & freshwater
wound infections fish. Infections often
involve aquatic
environments.
Pos indole
differentiates from
Salmonella.
Salmonella H2S, MR, motility, Lactose, indole, Found in poultry. May
lysine decarboxylase VP, PD, urease, be transmitted
(LDC) ONPG by reptiles. S. typhi
has Vi antigen, only
trace H2S, citrate neg.
Grouped by O antigens
(e.g., A, B, C),
serotyped by H
antigens (e.g., 1, 2).
Citrobacter freundii Nosocomial infections Gas, H2S, MR, citrate, VP, PD, LDC Lactose variable.
motility, ONPG ONPG & LDC
differentiate from
Salmonella.
Klebsiella pneumoniae Pneumonia, UTI, Lactose, gas, VP, H2S, indole, MR, Encapsulated.
septicemia citrate, urease (slow) PD, motility, Colonies usually
ornithine mucoid. Some strains
decarboxylase (ODC) hydrolyze urea slowly.
K. oxytoca is similar to
K. pneumoniae except
indole pos.
Motility & ODC
differentiate from
Enterobacter.

Enterobacter Opportunistic & Lactose, gas, VP, H2S, indole, MR, Colonies may be
aerogenes & cloacae nosocomial infections. citrate, motility, ODC PD mucoid. Same
UTI, RTI, & wound IMViC reactions as
infections Klebsiella
Serratia marcescens Opportunistic VP, citrate, motility Lactose, H2S, indole, Some produce red
pathogen. PD, urease pigment when
Pneumonia & incubated at RT.
septicemia
in immunosuppressed

Proteus vulgaris & UTI, wound infections, H2S, MR, PD, urease, Lactose Swarming. Burned
mirabilis septicemia motility chocolate odor.
P. mirabilis is most
common & indole
neg. P. vulgaris indole
pos, A/A on TSI
because of sucrose
fermentation
Morganella morganii Mainly nosocomial Indole, MR, PD, urease Lactose, H2S, VP,
infections. UTI, wound (weak), motility, ODC citrate
infections.

Providencia UTI, diarrhea Indole, MR, citrate, Lactose, H2S, VP P. rettgeri is urease
PD, motility pos.
Yersinia enterocolitica Diarrhea MR, urease Lactose, H2S, VP, GN coccobacilli.

17 | m a d e b y M C P M , R M T
 citrate, PD Bipolar staining.
Optimal temperature
25°–30°C. Motile
at 25°C but not 35°C.
CIN agar is selective.
Incubate 48 hr. Red
“bull’seye” colonies
surrounded by
colorless halo. Y.
pestis causes plague

SUMMARY OF KEY REACTIONS FOR ENTEROBACTERIACEAE

NON-MOTILE AT
LACTOSE NEGATIVE H2S (+) VP (+) PD (+) UREASE (+)
35C
Shigella
Edwardsiella
Salmonella
Citrobacter (some) Edwardsiella Proteus
Klebsiella Proteus Shigella
Serratia Salmonella Morganella
Enterobacter Morganella Klebsiella
Proteus Citrobacter Providencia
Serratia Providencia rettgeri Yersinia (motile at 22°C)
Morganella Proteus Klebsiella (slow)
Providencia
Yersinia

APPEARANCE OF ENTEROBACTERIACEAE ON SELECTED MEDIA

ORGANISM TSI MacCONKEY HEKTOEN ENTERIC XLD
Escherichia coli A/A, gas Flat, dry pink colony with Yellow Yellow
darker pink halo

Shigella K/A Colorless Green Colorless

Edwardsiella K/A, gas, H2S Colorless Colorless Red, yellow, or colorless with
or without black centers

Citrobacter A/A or K/A, gas, with Colorless at 24 hr. May Colorless Red, yellow, or colorless with
or without H become pink or without black centers
2S at 48 hr

Salmonella K/A, gas, H2S Colorless Green Red with black center

Klebsiella A/A, gas Pink, mucoid Yellow Yellow

Enterobacter A/A, gas Pink. May be mucoid Yellow Yellow

Serratia K/A Colorless at first, turning Colorless Yellow or colorless

pink. S. marcescens
may have red pigment at
RT

Proteus K/A (mirabilis) A/A Colorless. May swarm Colorless Yellow or colorless, with or
(vulgaris), gas, H2S without black centers

Morganella K/A, gas Colorless Colorless Red or colorless

Providencia K/A Colorless Colorless Yellow or colorless

Yersinia Yellow/orange Colorless to peach Salmon Yellow or colorless

DIARRHEAGENIC E. coli

18 | m a d e b y M C P M , R M T
 DISEASE GRAM STAIN OF
GROUP PATHOGENICITY TRANSMISSION OTHER
MECHANISM STOOL
Enterohemorrhagic Diarrhea, Undercooked Toxins (vertoxins RBCs but usually E. coli O157:H7 is
(EHEC). Also hemorrhagic meat, raw milk, or Shiga toxins) no polys most common
known as Shiga colitis, hemolytic apple cider isolate of group &
toxin–producing uremic syndrome pathogen most
(STEC) or Verotoxin (HUS). Most often isolated
-producing (VTEC) common cause of from bloody
renal failure in stools.
children in U.S. Non-O157 STEC
May be fatal, also causes
especially in young disease. DNA
or elderly probes can ID
genes that code
for toxins.
Report to public
health.
Enterotoxigenic ______________ Contaminated Toxins No polys or RBCs Profuse, watery
(ETEC) diarrhea in infants food or water stool. DNA probes
to detect toxins or
toxin genes
Enteroinvasive ____________ Contaminated Invasiveness Polys, RBCs,
(EIEC) Usually in young food or water mucus
children in
areas of poor
sanitation
Enteropathogenic Diarrhea in infants. Formula & food Adherence No polys or RBCs Watery diarrhea
(EPEC) Major pathogen in contaminated attachment with
infants in with mucus.
developing fecal material
Countries
Enteroaggregative Diarrhea in Nosocomial & Adherence Most labs can’t
(EAEC) Developing community attachment detect
countries. Chronic acquired
diarrhea in HIV-
infected patients
Diffusely adherent Diarrhea & UTI. Little known Adherence Not well studied.
(DAEC) Most common in about attachment
children in epidemiology
developing
countries

19 | m a d e b y M C P M , R M T
CHARACTERISTICS OF NON- FERMENTING GRAM NEGATIVE RODS
• Obligate aerobes. • Grow on SBA & CHOC in 24–48 hr
• Don’t ferment carbohydrates. K/K on TSI. • Most grow on MAC. Appear as non–lactose fermenter.
• May be oxidizers or nonoxidizers (asaccharolytic). • Resistant to variety of antibiotics.
• Most are oxidase pos. Differentiates from Enterobacteriaceae.
• Oxidation-fermentation (OF) medium: either open tube pos/closed tube neg (oxidizer) or
open tube neg/closed tube neg (nonoxidizer).
.
COMMONLY ISOLATED GRAM NEGATIVE RODS
ORGANISM PATHOGENICITY GRAM STAIN CULTURE KEY CHARACTERISTICS
Pseudomonas Usually not normal Long, thin, pale- On SBA: flat spreading Oxidase pos, catalase
aeruginosa flora. Important cause staining GNR. Slightly colonies. Usually beta pos, motile, grows at
of nosocomial pointed or hemolytic. Dull gray or 42°C. Grapelike odor.
infections, e.g., burn, rounded ends blue-green. Metallic Only nonfermenter to
wound, RTI, UTI, sheen. Grows on MAC produce pyocyanin.
bacteremia. & (4% don’t.)
Causes swimmer’s ear EMB (lactose neg). Also produces
& contact lens keratitis pyoverdin (fluorescent
pigment). Resistant to
many antibiotics.

20 | m a d e b y M C P M , R M T
Acinetobacter spp Part of normal flora of Pleomorphic GNCB in Can grow on most Oxidase neg
skin, pharynx in some. singles, pairs, short media, including MAC. (differentiates
Opportunistic chains. Can be Some produce from N. gonorrhoeae).
Pathogen. Nosocomial confused with N. purplish colonies Catalase pos. Non
infections, e.g., UTI, gonorrhoeae, (might be mistaken for motile. Resistant to
pneumonia, Moraxella. lactose fermenter). many antibiotics.
Septicemia, May retain crystal
meningitis. 2nd to P. violet in broths &
aeruginosa in direct smears & be
frequency. A. confused with GPC
baumannii is most
common
Stenotrophomonas Not part of normal Straight or slightly On SBA: large, Oxidase neg. Catalase
maltophilia flora. curved slender GNR in nonhemolytic. May be pos.
Colonizes singles or pairs. light Motile. Rapid
immunocompromised yellow. Agar may have oxidation of
& cystic fibrosis lavender-green maltose, weaker
patients. Common in discoloration in areas oxidation of
hospital. Nosocomial of heavy glucose. Ammonia
infections, growth. Grows on odor. Resistant to
e.g., pneumonia MAC many antibiotics. Disk
diffusion can give false
results.
Broth dilution
recommended

CAMPYLOBACTER AND HELICOBACTER

ORGANISM PATHOGENICITY GRAM STAIN CULTURE KEY CHARACTERISTICS
Campylobacter Most common cause Curved, slender, Microaerophilic & Darting corkscrew
jejuni of bacterial diarrhea. GNR. capnophilic. Grown on motility. Oxidase,
Sources of infection: “Seagulls,” loose Campy-BAP at 42°C in catalase, & hippurate
chickens, raw milk, spirals, & S ↑CO2. (Can grow at hydrolysis pos.
pets shaped. 37°C; normal enteric flora
Stain faintly. inhibited by 42°C
incubation.) Slow
growing. Hold plates 3
days.

Campylobacter coli Similar to C. jejuni, Same as above. Same as above Rarely differentiated
but less severe. from C. jejuni. Hippurate
Usually foodborne hydrolysis neg.
Campylobacter Causes bacteremia in Same as above. Most often isolated in Oxidase & catalase pos.
fetus immunocompromised blood cultures. Hold for 2 Hippurate hydrolysis neg.
& elderly. wk. Inhibited on Campy
Uncommon stool agar. Grows on routine
isolate media at 37°C, not at
42°C.

Helicobacter pylori Gastritis, duodenal & Curved, slender, Grows on nonselective Rarely cultured. Rapid
peptic ulcers. Possible GNR media incubated at 37°C urease tests on gastric

21 | m a d e b y M C P M , R M T
 risk factor for in same atmosphere as biopsy, urea breath test,
gastric carcinoma Campy. Doesn’t grow at histology, PCR, serology.
42°C. Slow growing

VIBRIO AND RELATED ORGANISMS

ORGANISM PATHOGENICITY GRAM STAIN CULTURE KEY
CHARACTERISTICS
Vibrio cholerae Cholera (acute Diarrhea, Small comma-shaped Nonhalophilic (doesn’t “Rice water” stools.
dehydration, electrolyte GNR in direct smears; require NaCl for Oxidase pos. Motile.
imbalance). Transmitted straight pleomorphic growth). Grows on Serological ID with
by contaminated water, GNR in culture. SBA, CHOC, antisera to O ag. O1 &
seafood. Uncommon in MAC (NLF). Large O139 strains cause
U.S., but may be seen in yellow colonies on epidemics. El Tor
coastal areas. TCBS (ferments biotype causes most
sucrose). Alkaline cases worldwide.
peptone water (APW) Notify public health
can be used for department,
enrichment. send for confirmation.

Vibrio vulnificus 2nd most serious type of Straight or curved Halophilic (salt loving, Oxidase pos. Motile
vibrio infection. GNR requires addition of
Immunocompromised or Na+). Most are green
individuals with liver on TCBS; some are
disease: septicemia yellow. May look like
following consumption of enteric on MAC
raw oysters, water- because some are
associated wound lactose pos.
infections. Healthy
individuals:
Gastroenteritis. Seen in
U.S.
Vibrio 2nd most common Vibrio Straight or curved Requires 1% NaCl for Oxidase pos. Motile.
Parahaemolyticus to cause gastroenteritis. GNR. growth. Grows on
Contaminated seafood. SBA, MAC (NLF). Blue-
Waterassociated wound green colonies on
infections. Seen in U.S. TCBS (doesn’t
ferment sucrose).

Aeromonas spp Gastroenteritis & wound Straight or curved Grows on routine Oxidase pos
infections, often related GNR media. Most are beta (differentiates from
to aquatic exposure. hemolytic on SBA, NLF Enterobacteriaceae).
Septicemia, meningitis. on MAC. Doesn’t grow Motile
on TCBS. CIN & APW
can be used for
selective isolation
Plesiomonas Gastroenteritis from Pleomorphic GNR in Grows on SBA, CHOC. Biochemical &
shigelloides contaminated water or singles, pairs, short Most grow on MAC, antigenic similarities
(now included in seafood. Bacteremia & chains, or long appear as NLF. Doesn’t to Shigella.
Enterobacteriaceae) meningitis in immune- filaments grow on TCBS. Oxidase pos. Motile.
compromised & neonates

22 | m a d e b y M C P M , R M T
HAEMOPHILUS
ORGANISM PATHOGENICITY GRAM STAIN CULTURE KEY CHARACTERISTICS
H. influenzae Normal flora of upper Small, pleomorphic Grows on CHOC in Should be serotyped.
respiratory tract. GNCB to long 5%–10% CO 2.
Causes sinusitis, filaments. Capsules Translucent, moist,
otitis media, may be seen. tannish colonies.
pneumonia, Encapsulated strains
bronchitis, often in form larger & more
elderly or mucoid colonies.
compromised. Type b Mousy or bleach-like
common cause of odor. May

23 | m a d e b y M C P M , R M T
 pneumonia & demonstrate
meningitis in children satellitism with staph
where Hib vaccine not on SBA.
available.

H. influenzae biotype Both cause Same as above Same as above Characteristics are
aegyptius & H. conjunctivitis similar to H.
aegyptius (pink eye). H. influenzae.
influenzae biotype Difficult to
aegyptius also causes differentiate
Brazilian purpuric
fever

H. parainfluenzae, Normal flora of upper Small, pleomorphic Colonies are larger,

H. haemolyticus, & respiratory tract. Low GNCB to long dry, & tannish.
H. parahaemolyticus incidence of filaments.
pathogenicity.

H. ducreyi Never normal flora. Small GNCB, bipolar Difficult to culture. Can ID by PCR
Causes chancroid staining. May
(sexually transmitted resemble schools of
disease). fish or railroad tracks

DIFFERENTIATE HAEMOPHILUS SPECIES

Note that porphyrin/ALA reactions & X factor requirement are opposite, i.e., species that are pos for porphyrin/ALA don’t
require X. Species with “para” in name only require V factor & are porphyrin/ALA
pos. Species with “haemolyticus” in name are hemolytic on rabbit & horse blood HEMOLYSIS
agar. Multitest biochemical systems available for ID ON RABBIT
AND
SPECIES
HORSE
BLOOD
AGAR
H. influenzae and
0
H. aegyptius
H. parainfluenzae 0
H. haemolyticus +
H. parahaemolyticus +
H. ducreyi 0
MISCELLANEOUS GRAM NEGATIVE RODS
ORGANISM PATHOGENICITY GRAM STAIN CULTURE KEY CHARACTERISTICS
Bordetella pertussis Whooping cough in Small GNCB Grows on Bordet- Fluorescent antibody
children & adults Gengou & Regan-Lowe stain. DTaP vaccine
(charcoal, (diphtheria, tetanus,
horse’s blood) after 3– pertussis).
7 days

______________ Trench fever, relapsing Intracellular GNCB Not practical. Takes 9– Dx usually by
fever, bacteremia, 40 days serological or to
endocarditis, grow.molecular
cat-scratch disease methods.

24 | m a d e b y M C P M , R M T
Brucella Brucellosis (undulant Tiny, faintly staining Most often isolated Oxidase & urease pos.
fever). From GNCB from blood cultures or Level 3 pathogen.
unpasteurized milk or biopsies of reticu- Potential bioterrorism
contact with infected loendothelial (RE) agent. Re- portable
goats, cows, hogs, tissue. Blood cultures disease. Serological
dogs incubated in ↑ CO2 tests are primary
for 3 wk. Culture not means
sensitive of Dx.

Francisella Tularemia (rabbit Pale-staining, small, Grows on special Level 3 pathogen.

fever). Man infected pleomorphic, intra- media en- riched with Direct fluorescent ab
by tick or handling cellular GNCB with glucose & cystine (e.g., methods for ID. Send
infected animal bipolar staining blood cysteine glucose to public health lab for
agar), TM, & BCYE. confirmation.
Small, transparent Serological tests.
colonies after
3 days.
Gardnerella Normal flora of female Small, pleomorphic Not recommended for Catalase & oxidase
genital tract. gram-variable rods Dx of BV. Grows on neg. Tests for Dx of BV:
Associated with SBA, PEA, CNA, clue cells (vaginal
bacterial vaginosis human blood Tween epithelial cells covered
(BV) when it & (HBT) agar, V agar. with gram-variable
anaerobic GNR are Produces diffuse beta rods), whiff test (10%
predominant & hemolysis only on KOH added to vaginal
Lactobacillus is absent. media containing secretion
Linked to maternal & human blood.  fish-like odor).
neonatal infections, Requires ↑ CO2 & 48–
bac- teremia, rarely 72 hr incubation.
UTI

Legionella Legionnaire’s disease, Small, pleomorphic, Grows on BCYE in 3–4 Oxidase pos. ID by
Pontiac weakly staining GNR days. immunofluorescent
fever. Found in water Pale yellow-green stain. Serology.
(e.g., air fluorescence with
conditioners, Wood’s lamp.
showerheads,
whirlpools).

Pasteurella Contracted by Pleomorphic GNCB Grows on SBA & Oxidase, catalase, &
multocida inhalation of aerosol with bipolar staining CHOC, but not indole pos
Contracted by bite or MAC. Musty odor.
scratch
of cat or dog or
contact with
infected carcass.
Wound &
respiratory tract
infection

25 | m a d e b y M C P M , R M T
Kingella kingae colonizes the upper respiratory tract and is primarily associated with infections of bones and joints, as well
as endocarditis in children and young adults. It grows on sheep blood agar and is β-hemolytic. A Gram’s stain shows short,
plump, gram-negative rods with square ends. Other less pathogenic species in the genus are K. denitrificans and K. oralis.

Capnocytophaga is normal oral flora in humans. It may cause serious infections in immunosuppressed patients. It has been
associated with dog bites and is usually isolated from blood or cerebrospinal fluid. It grows on sheep blood agar, but is
capnophilic and must have CO2 for growth. Colonies are beige or yellow and show a haze of growth at the periphery as a
result of gliding motility. Gram’s stain shows fusiform, gram-negative bacillus

Cardiobacterium hominis is a pleomorphic, gram-negative rod that grows on blood, but not MacConkey agar. It is normal
human oral flora and is found in patients who have endocarditis and bacteremia. The colonies are small, and growth is slow.

SPECIMENS FOR ANAEROBIC CULTURE

ACCEPTABLE UNACCEPTABLE
Bile Expectorated sputum
Blood Feces
Body fluids Gastric juice
Bone marrow Swabs
Percutaneous lung aspirate or biopsy Voided or catheterized urine
Suprapubic bladder aspirates Bronchial washings (unless obtained with a double-
Tissue lumen plugged catheter)
Transtracheal aspirate
Wound

QUALITY CONTROL FOR ANAEROBIC ENVIRONMENT

Methylene blue strip : Blue = O2 present, white or colorless = no O2.
Resazurin : Pink = O2 present, colorless = no O2.
Oxygen analyzers : More expensive. Real-time monitoring

CULTURE MEDIA FOR ANAEROBES

MEDIUM USE
Nonselective enrichment medium that grows obligate & facultative anaerobes. Contains
Anaerobic blood agar (CDC)
yeast extract, L-cysteine, hemin, & vitamin K.

Bacteroides bile–esculin Selective differential medium for Bacteroides fragilis. Bile salts & gentamicin act as inhibitors.
(BBE) agar B. fragilis colonies black with dark halos due to esculin hydrolysis.

Brucella blood agar Enriched medium that grows obligate & facultative anaerobes.
Colistin–nalidixic acid (CNA) Selective medium that grows obligate anaerobes & GP facultative anaerobes.
blood agar

Cycloserine cefoxitin Selective & differential for Clostridium difficile. Colonies are yellow due to fermentation of
fructose egg yolk (CCFA) fructose. Chartreuse fluorescence.
agar
Egg-yolk agar (EYA) For determination of lecithinase & lipase production by clostridia & fusobacteria.

Kanamycin-vancomycin Also known as laked blood kanamycin-vancomycin (LKV) agar. Most commonly used selective
laked blood medium for isolation of anaerobic GNRs, especially Bacteroides & Prevotella

26 | m a d e b y M C P M , R M T
 (KVLB) agar
Phenylethyl alcohol (PEA) Selective medium that inhibits enteric GNRs & grows obligate anaerobes & GP facultative
agar anaerobes.

All-purpose medium that supports growth of most aerobes & anaerobes. Can be used as
backup broth to detect organisms present in small numbers or anaerobes. Thioglycolate acts
Thioglycolate (THIO) broth
as reducing agent. Aerobes grow at top, strict anaerobes at bottom, facultative anaerobes
throughout. Store at RT. Boil & cool before use

ANAEROBIC GRAM POSITIVE COCCI

ORGANISM PATHOGENICITY GRAM STAIN OTHER
Finegoldia magna (formerly Normal flora on skin & in GPC in singles, pairs, tetrads, Small colonies. May take 48
peptostreptococcus mouth, intestines, female clusters. Resembles staph. hr to grow.
magnus) genital tract. Most Catalase & indole neg.
commonly isolated & most Resistant to SPS.
pathogenic anaerobic GPC.
Associated with skin
infections, decubitus
ulcers, septic arthritis, bone
infection following
orthopedic surgery, oral &
female genital tract
infections, bacteremia.

Peptostreptococcus
anaerobius
Peptoniphilus
Asaccharolyticus (formerly
Peptostreptococcus
asaccharolyticus)
Actinomyces
Normal on skin & in mouth,
GI, & GU tracts. Mixed
infections of skin, soft
tissues, GI tract, female
genital tract, bones, joints,
lungs, brain
Normal on skin & in GI & GU
tracts. Associated with
obstetric & gynecological
infections
Infects brain, face, lungs,
genitals
Tiny GPC in chains.
Resembles strep
GPC in pairs, short chains,
tetrads, clusters
Short or long GPR. Branched
or unbranched. Banded
appearance. Can break into
club-shaped rods resembling
diphtheroids.
Small gray-white colonies in
24-48 hr. Sweet odor.
Sensitive to SPS
Yellow colonies. Musty odor.
Resistant to SPS.
Indole pos.
Sulfur granules may be seen
in discharge. Crush & stain to
reveal characteristic Gram-
stain morphology. “Molar
tooth” colonies.

Clostridium botulinum
Clostridium difficile
Botulism due to ingestion of
toxin in inadequately cooked
or improperly canned foods.
Infant botulism due to
ingestion of spores in honey.
Wound botulism from
injection drug use
Antibiotic-associated
diarrhea, pseudo-
membranous colitis
GPR with oval subterminal
spores
Thin GPR. May form chains.
Rare oval subterminal
spores.
Reportable disease. Toxin
testing at public health labs
Yellow ground-glass colonies
on cycloserine cefoxitin
fructose agar (CCFA). Usually
not cultured. Must
demonstrate toxin
production. Toxins A & B.
Tissue culture or EIA.

Clostridium perfringens
Clostridium tetani
Normal in GI tract. Causes
gas gangrene, food
poisoning
Causes tetanus. Deep
Large GPR with blunt ends in
chains. “Box cars.” Tendency
to stain gram neg. Usually no
spores seen.
GPR with swollen terminal
Most commonly isolated
Clostridium. Double zone of
beta hemolysis on SBA
Rare in U.S. because of DTaP

27 | m a d e b y M C P M , R M T
 wounds infected with soil spores. “Drumstick.” “Tennis vaccine. Lab confirmation
racquet.” Becomes gram neg rarely required.
after 24 hr.

Propionibacterium Normal skin flora. Common Club-shaped, pleomorphic Most common anaerobic
contaminant of blood GPR. Diphtheroid-like. GPR. Catalase & indole pos.
cultures. Causes acne;
infections associated with
artificial joints, catheters,
shunts, artificial heart valves;
keratitis; bacteremia;
endocarditis

Lactobacillus Normal in mouth, GI tract. Pleomorphic, long, thin, Aerotolerant anaerobes.

Predominant flora of vagina non–spore-forming GPR Grow better under anaerobic
during reproductive years. often in chains. Chains of conditions. Colonies vary
Produces lactic acid, rods in THIO greatly. May resemble S.
maintains vaginal pH. viridans. Catalase neg.
Absence in vagina
predisposes to bacterial
vaginosis & yeast infections.
Rare cause of endocarditis,
septicemia in
immunocompromised

GRAM NEGATIVE ANAEROBES

ORGANISM PATHOGENICITY GRAM STAIN OTHER
Gram Negative Cocci
Veillonella Normal flora of upper Tiny GNDC in pairs, clusters, May show weak red
respiratory tract, GI & GU short chains. Can resemble fluorescence under
tracts. Usually in mixed Neisseria UV light. Doesn’t grow on
culture. Can cause infections KVLB. Usually doesn’t reduce
in immunocompromised nitrates
Gram Negative Rods
Bacteroides fragilis Mixed infections below Pleomorphic, pale, Most common anaerobe
diaphragm irregularly staining GNR isolated. Resistant to bile.
Hydrolyses esculin.
Turns BBE brown.

Fusobacterium Pulmonary infections, brain GNR. F. nucleatum is long & Indole pos.
abscesses, oral lesions thin with tapered ends Most fluoresce chartreuse
(spindle shaped) (green-yellow) under UV
light.

28 | m a d e b y M C P M , R M T
Porphyromonas Head, neck, & pleuro- GNCB Slow growing. Brown to
pulmonary infections black on SBA. Some
fluoresce brick-red to orange
under UV light. Doesn’t grow
on KVLB
Prevotella Head, neck, & GNCB Slow growing. Some are
pleuropulmonary pigmented. Brown to black
infections on SBA & KVLB. Some
fluoresce brick-red to orange
under UV light. Doesn’t grow
on BBE

LABORATORY IDENTIFICATION OF MYCOBACTERIA

Mycobacterium tuberculosis transmitted by inhalation of aerosols. Low infective dose.
Separate room, if possible, with non-recirculating ventilation system & negative air
pressure. BS-2 practices. Use of BSC for all procedures that might generate aerosols.
Lab safety Electric incinerators instead of flames to sterilize wire loops. Slide-warming trays
instead of flames to fix slides. Tuberculocidal disinfectants to clean equipment & BSC,
e.g., 1:10 dilution of household bleach made fresh daily. UV light in BSC (only when
not in use). Annual testing of employees for exposure, e.g., Mantoux skin test with PPD.

29 | m a d e b y M C P M , R M T
Specimens requiring
Sputum & other specimens with normal flora such as gastric lavage, urine, feces
digestion/decontamination
Specimens not requiring
Tissue or body fluids collected aseptically.
digestion/decontamination

Digestion/decontamination Sodium hydroxide (NaOH), N-acetyl-L-cysteine (NALC)/NaOH, benzalkonium chloride,

methods oxalic acid. NaOH is digestant & decontaminating agent. NALC is liquefying agent.

Cell walls have high lipid content (mycolic acids). Difficult to stain. Resist decolorization
Staining by acid alcohol (acid-fast). Gram stain poorly. Use carbolfuchsin or fluorochrome acid-
fast stains.

Agar-based (Middlebrook 7H10 & 7H11), egg-based (Löwenstein-Jensen, Petragani,

American Thoracic Society), liquid (Middlebrook 7H9). Combination of a solid-based
Media
medium & a liquid-based medium recommended for primary isolation

Incubation 35°C in 5%–10% CO2. (Exception: If M. marinum, M. ulcerans, or M. haemophilum

suspected, incubate at 25°–30°C.)

Rate of growth Slow. On solid media, most require 2–6 wk incubation; rapid growers 2–3 days.
More rapid growth in liquid media
Liquid broth inoculated, placed in blood culture instrument for automatic or continuous
Automated systems for
monitoring. Growth indicated by consumption of O2 or production of CO2. Earlier
recovery
detection than manual methods.

Colony morphology, growth rate, optimum temp for growth, photoreactivity,

Methods for identification biochemical tests, chromatography, nucleic acid hybridization, PCR, automated DNA
sequencing.

ACID FAST STAINS

STAIN PRIMARY STAIN DECOLORIZE COUNTERSTAI APPEARANCE OF AFB OTHER
R N
Ziehl-Neelsen Carbolfuchsin Acid alcohol Methylene blue Red, slightly curved, Requires heat. Examine at
beaded rods (2–8 least 300 OI fields. Wipe
μm). Blue lens after pos smears to
Background avoid cross-contamination
. & false pos.

Kinyoun Carbolfuchsin Acid alcohol Methylene blue Red, slightly curved, Cold stain. Examine at least
beaded rods (2–8 300 OI fields. Wipe lens
μm). Blue background after pos smears to avoid
cross-contamination &
false pos.

Fluorochrome Auramine- Acid alcohol Potassium Yellow-orange rods More sensitive than
Rhodamine permanganate against dark carbolfuchsin. Faster to read.
or acridine background Can examine at 250×.
orange Read immediately or store
at 2°C–8°C in dark to avoid
fading. Examine at least
300 fields. Pos should be
confirmed with
carbolfuchsin stain. Rapid
growers might not stain.

CLASSIFICATION OF MYCOBACTERIA BASED ON PATHOGENICITY

GROUP PATHOGENICITY SPECIES
Mycobacterium tuberculosis Cause human tuberculosis (TB). M. tuberculosis, M. bovis, M.
complex (“tubercle bacilli”) africanum, M. microti, M. canet
30 | m a d e b y M C P M , R M T
Atypical mycobacteria, Some cause pulmonary infection similar
Non-tuberculous mycobacteria to TB but not transmitted person to
(NTM), or mycobacteria other person. Usually in immuno-
than tubercle (MOTT) bacilli compromised. Contracted from
environment (soil, water)
CLASSIFICATION OF NON- TUBERCULOUS MYCOBACTERIA BASED ON PHYSIOLOGY
RUNYOUN (WHY?) GROUP CHARACTERISTICS
Yellow pigment when exposed to light
Photochromogens after being grown in dark.
>7 days to appear on solid media.
Yellow pigment when grown in light or
Scotochromogens (ORIGIN OF THE
dark.
WORD?)
>7 days to appear on solid media.
No pigment produced in light or dark.
Nonphotochromogens
>7 days to appear on solid media.
Rapid growers <7 days to appear on solid media
MEDICALLY IMPORTANT MYCOBACTERIA
ORGANISM PATHOGENICITY
M. tuberculosis Tuberculosis
GI infection following consumption of unpasteurized
M. bovis
milk
Causes lung infection. Disseminated disease in
M. africanum
immunocompromised
Most common NTM to cause lung disease in U.S.
M. avium complex (MAC)
Most common systemic bacterial infection in AIDS
patients. Cervical lymphadenitis in children
31 | m a d e b y M C P M , R M T
M. avium, M. intracellulare, M. kansasii,
M. malmoense,
M. chelonae, M. xenopi, M. gordonae
EXAMPLES
M. marinum, M. kansasii
M. gordonae
M. avium complex, M. ulcerans
M. smegmatis
KEY POINTS
Slow growth. Average recovery time by
conventional methods 21 days.
Rough, dry, buff-colored (no
Pigmentation) colonies. Serpentine
cording on smear from culture.
Niacin & nitrate pos.
68°C catalase neg.
Rare in U.S. Bacille Calmette-Guérin
(BCG) is attenuated strain used in
vaccine in some parts of world.
Causes false-pos PPD test (WHY)
Spread by respiratory droplets.
Endemic in equatorial
Africa. Rare in U.S
M. avium & M. intracellulare. Usually
not differentiated. Contracted from
environment. Non photochromogens.
 Skin, joint, bone, lung infections in
M. hemophilum
immunocompromised. Lymphadenitis in children
Chronic infection of skin & Subcutaneous tissue.
M. ulcerans
Buruli ulcers
2nd most common NTM to cause lung disease. Causes
M. kansasii skin & soft tissue infections, lymphadenitis. Can
disseminate in immunocompromised
Requires hemin for growth. Grows best
at 30°C. Non photochromogen
Endemic in tropics. 3rd most common
mycobacterial disease after TB &
leprosy. Grows best at 30°C.
Nonphotochromogen
Photochromogen. Can ID by 16sRNA
probe.

Contracted from swimming pools,

M. marinum Skin infections
aquariums. Grows best at 30°C.
Photochromogen
Common cause of cervical lymphadenitis in children
M. scrofulaceum Scotochromogen.
in Africa
“Tap-water bacillus.” Laboratory
M. gordonae Rarely causes infection contaminant. Scotochromogen. Can ID
by PCR & nucleic acid probes.

In water, soil, dust. Contaminant of medical devices.

Skin & soft tissue infections. Lung infections in those
M. abscessus with chronic lung disease, e.g., cystic fibrosis. Tap water is reservoir. Rapid grower.
Disseminated infection in
immunocompromised

Disseminated cutaneous infections in

M. chelonae immunocompromised, infections of lungs, bone, Rapid grower
central nervous system, prosthetic heart valves
Common in environment. Rapid grower.
Weakly gram pos.
Infections of skin, soft tissues, IV & injection sites,
M. fortuitum Weakly acid fast. Stains with
surgical wounds
carbolfuchsin, but may not with
fluorescent stain.

Endemic in Southern hemisphere. <100

cases per year in U.S. Most in TX, CA,
LA, HI, PR. Armadillos may be
reservoir. Doesn’t grow on artificial
M. leprae Leprosy (Hansen’s disease)
media. Can be grown in footpads of
mice & armadillos. Dx by acid-fast
stain of tissue. Less acid fast than M.
tuberculosis

32 | m a d e b y M C P M , R M T
CHLAMYDIA AND CHLAMYDOPHILA
Members of the family Chlamydiaceae are obligate intracellular parasites. The three most common species are Chlamydia
trachomatis, Chlamydophila pneumoniae (previously Chlamydia pneumoniae), and Chlamydophila psittaci (previously
Chlamydia psittaci). There are two forms in the growth cycle of the organism: the elementary body is the infectious form, and
the noninfectious reticulate body is the intracellular reproductive form
ORGANISM PATHOGENICITY
Most common sexually
transmitted bacterial
infection in U.S. Trachoma,
lymphogranuloma
Chlamydia trachomatis venereum, nongonococcal
urethritis, pelvic
Inflammatory disease.
Pneumonia & conjunctivitis
in newborns.
Chlamydophila (Chlamydia) 3rd most common cause of
pneumoniae acute RTI.
Psittacosis (“parrot fever”).
Chlamydophila (Chlamydia)
Rare in U.S
psittaci
(ORNITHOSIS IN HUMAN?)
SPIROCHETES
Three genera in the order Spirochaetales cause human disease: Treponema, Leptospira, and Borrelia. These bacteria are
characterized by their helical shape with spiral coils.
ORGANISM PATHOGENICITY
Borrelia recurrentis Relapsing fever
Lyme disease (erythema
chronicum migrans,
Borrelia burgdorferi
neurological & cardiac
abnormalities, arthritis)
Treponema pallidum
Syphilis
subspecies pallidum
Weil’s disease (infectious
Leptospira interrogans
jaundice, leptospirosis)
33 | m a d e b y M C P M , R M T
LAB DX
Giemsa stain, direct
fluorescent antibody stain,
cell culture, rapid antigen
assay (EIA), nucleic acid
amplification tests (NAATs),
DNA probes, serological tests
for antibodies
Serological tests are method
of choice.
Serological tests
LAB DX
Giemsa- or Wright-stained
blood smears.
Grows on modified Kelly
medium. Serology is most
common method. Doesn’t
grow on artificial media.
Darkfield microscopy,
fluorescent stain. Serology is
most common method.
Culture.
Growth = turbidity below
surface of semisolid
Fletcher’s medium.
Hold cultures for 6 wk.
Serological tests, PCR
OTHER
Obligate intracellular
parasite. Need epithelial
cells for culture.
Wooden swabs are toxic.
Cell culture was gold
standard; NAAT now
preferred for genital
specimens. Commercial
systems available for
simultaneous detection
of N. gonorrhoeae.
Obligate intracellular
parasite. Risk factor for
Guillain-Barre syndrome.
May be risk factor for
cardiovascular disease.
Obligate intracellular
parasite. Spread by birds
OTHER
Transmitted by ticks & lice.
Most commonly reported
tick-borne infection in U.S.
Vector = Ixodes tick.
Worldwide.
Zoonotic disease.
Transmitted by urine
of infected animal. Organism
in blood & CSF during first
week, then urine
MYCOPLASMA/ UREAPLASMA
The organisms in the family Mycoplasmataceae are small, free-living organisms that lack a cell wall. The three most common
isolates are Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum.

ORGANISM PATHOGENICITY OTHER

Primary atypical pneumonia
Mycoplasma pneumoniae
(walking pneumonia)
Smallest free-living cells. Lack cell wall.
Pleomorphic. Not visible by Gram
staining. Difficult to grow. Culture rarely
performed. May ake 21 days or more.
Usually Dx by serology.

Only species that will grow on SBA &

CHOC but may require 4 days. Pinpoint
translucent colonies easily overlooked.
Mycoplasma hominis Urogenital tract disease “Fried-egg” colonies seen with
stereomicroscope after staining with
methylene blue. Serological methods
available.

Granular brown appearance on A8 agar

Ureaplasma Urogenital tract disease because of urease production.
Serological methods available.

RICKETTSIA
GENUS PATHOGENICITY
Rickettsia Rocky Mountain spotted
fever, rickettsial pox,
epidemic typhus, murine
typhus, scrub typhus
LABORATORY DIAGNOSIS
Don’t grow on artificial
media. Grow in lice, ticks,
tissue culture, eggs. Usually
Dx by serology.
Immunohistologic &
molecular methods
available.
OTHER
Obligate intracellular
parasite. Transmitted by
ticks, mites, lice, fleas. Rocky
Mountain spotted fever is
most common rickettsial
infection in U.S. Caused by R.
rickettsii. BSL-3 biohazard.

Coxiella Q fever Grows in cell culture. Usual Obligate intracellular

Dx by serology. parasite. Zoonotic
Immunohistologic & disease. Found in cattle,
molecular methods sheep, goats. Highly
available. contagious. BSL-3 biohazard.
Reportable disease.
Potential bioterrorism
agent.

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Ehrlichia Ehrlichiosis Morulae (clusters of
organisms
that resemble blackberry) in
WBCs. Usual Dx by serology
Obligate intracellular
parasite. Infects WBCs.
Transmitted by ticks. Disease
similar to Rocky Mountain
spotted fever

FECAL PATHOGENS

OXIDASE (+) OXIDASE (-)

VIBRIO E. coli
CAMPYLOBACTER SHIGELLA
AEROMONAS SALMONELLA
PLESIOMONAS YERSINIA

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