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Laboratory Diagnosis

and Bacterial
Identification
Dr. Drummelsmith
Fundamentals 2
How do you find me?
• Faculty offices
• jdrummelsmith@rossu.edu
• Make appt – tell me when you’re available (3 times)
• Practice questions
– Workshop materials (on Canvas)
– Handout learning activities (on Canvas)
– Quizzes (on Canvas)
– Enrichment Sessions (live) and PMW (live and Canvas)
– Access Medicine (HUNDREDS of questions)
– UWorld subscription (useful once you start organ systems)
– Becker QMD (once you’re doing well with UWorld)
Learning Objectives
1. Describe how clinical data are important in the identification of the causative organism/agent of an infection and
how laboratory tests play a role in diagnosis and treatment.
2. Describe the important principles for proper specimen collection and processing.
3. Describe how specimens are processed in the lab. For each test described, identify when in the process that
result would be obtained.
4. Recognize whether members of a genus are part of the normal microbiota, and list the anatomic site(s) at which
each is most likely found.
5. Describe and differentiate the types of microscopy useful in laboratory diagnosis, and for what each is used.
6. Describe the principles and methods used for the cultivation of various microbes.
1. Explain how each medium in the given table is selective and/or differential.
2. Name the organisms these media select for and/or differentiate and describe their resulting appearance.
3. Describe the principles used for the cultivation of obligate intracellular organisms including viruses.
7. Explain the principles of the following biochemical tests used in the identification of bacteria isolated in culture:
catalase, oxidase, coagulase, urease, carbohydrate fermentation, H2S detection, esculin production, bile solubility,
thioglycollate, hemolysis.
8. Explain how detection of microbial antigens and nucleic acids can be used to diagnose an infectious disease.
1. Explain how antibody agglutination, sandwich ELISA, and chromatographic immunoassay techniques work
and when they can/should be used.
2. Explain how microbial nucleic acid detection methods work, and when they are used.
9. Explain how infectious diseases are diagnosed via detection of pathogen-specific antibodies from patient serum.
1. Recognize when and for what types of microbes detecting antibodies is a useful tool.
2. Discuss the advantages and limitations of serological techniques.
10. Differentiate microbial pathogens based on key characteristics. Use the TESTABLE BACTERIA list for guidance.
LOs: How to work with the lab
1. Describe how clinical data are important in the identification
of the causative organism/agent of an infection and how
laboratory tests play a role in diagnosis and treatment.
2. Describe the important principles for proper specimen
collection and processing.
3. Describe how specimens are processed in the lab. For each
test described, identify when in the process that result would
be obtained.
4. Recognize whether members of a genus are part of the
normal microbiota, and list the anatomic site(s) at which each
is most likely found.
Role of the Clinical Microbiology
Laboratory in Diagnosing Infections
• Provide accurate information regarding:
– Presence of microorganisms in a specimen
– Characterization/ID of the infectious agent
– Antimicrobial susceptibility, if possible/relevant
• Ability to achieve these goals is limited by:
– Quality of the specimen
– Specimen transportation
– Availability, sensitivity, specificity of diagnostic
techniques
The path from bedside to benchtop
and back
Specimen Specimen
Patient
collected Transported

Result Specimen
reported to Test Performed Received and
physician Processed

Physician Physician
interprets relates result
result to patient care
Specimen collection and processing
Minimize • From actual site of infection
• Minimally contaminated with material from adjacent
Contamination tissues, organs or secretions
• Example: smears for malaria diagnosis should be
Appropriately collected at different times during the day because
parasitemia can be intermittent
Timed • Obtained before administering antibiotics

In the Appropriate • Follow guidelines
Container/Device
• Insufficient volumes of specimens can compromise
Sufficient Quantity the validity of results
• Patient name
• ID number
Properly labeled • Specimen source
• Ordering physician
• Date/hour collected
Where are the normal microbiota?
• A knowledge of normal microbiota and
contaminants guides proper specimen
handling and interpretation of results
Anatomic sites normally Anatomic sites with normal
sterile microbiota
Blood Mouth, nares
Bone marrow Upper respiratory tract
Cerebrospinal fluid Skin
Serous fluids Gastrointestinal tract
Tissues Female genital tract
Lower respiratory tract Urethra
Bladder Table from Mims Medical Microbiology, 4th ed., p.462
Types of Specimens
• Direct from sterile site
– Suprapubic needle specimen of urine,
bronchoalveolar lavage fluid
– All organisms significant
• From sterile site through site w/ normal
microbiota
– Midstream urine specimen, sputum
– Only pathogens are significant
• Sites with normal microbiota
– Oropharynx, GI tract, skin, female genital tract,
urethra
– Many organisms, some of pathogenic genera…are
they significant?
Basic Flow of Laboratory Diagnosis
• Direct examination of patient specimens for presence of
etiologic agents
• Results same day; “preliminary report”
• Microscopy, rapid antigen tests, nucleic acid tests, serological
techniques; some methods may provide definitive diagnosis
• Growth and cultivation (culture) of the causative agent(s)
from the same specimens
• By day 2-3, some identifying info available; “interim report”
• Growth characteristics, staining, rapid biochemical or antigen tests
designed to be carried out on isolated bacteria
• Analysis of the cultured agent(s) to establish identification
and other characteristics
• Definitive ID/susceptibilities can take a few more days; “final report”
• Microscopy, biochemical, antigen, nucleic acid, mass spectrometry,
antibiotic susceptibilities
Lab results to treatment
Infection suspected: obtain
Primary prophylaxis samples for microbiologic
analysis

Preliminary and
Empiric treatment
interim microbiology
24-48 hours
results available
(e.g. Gram stain)
Targeted treatment
24-48 hours
Final
microbiology Definitive treatment
results available
Days - months
“Cure”
Secondary prophylaxis
Weeks to years
How are specimens processed?
Sputum: http://www.mayomedicallaboratories.com/test-catalog/Overview/8095

Urine, sputum, stool, etc. Blood

• Microscopy • Inoculated into bottles for
• Gram stain for most enrichment
• Specific techniques e.g. ova and parasite • Signal when growth occurs
(O+P) trichrome stain for stool, acid-fast
stain if AFB suspected (sputum, CSF) • Then stained, cultured
• Directly cultured onto media
Microbial identification
• Based on properties of an organism
– Morphology, metabolism, products
• Process designed to produce a combination of
results unique to a particular pathogen
– Could be as “simple” as microscopy of an
organism with a unique appearance, a PCR result,
or a protein fingerprint
– Could require multistep process, using
microscopy, growth characteristics, and
biochemical tests
Culture-Independent Diagnostic Tests
(CIDTs)
Detection of Infectious Agents

Direct microscopic examination (visualization)

Detection of a specific microbial product

Detection of specific antibodies against a
particular pathogen
LOs: Microscopy
5. Describe and differentiate the types of
microscopy useful in laboratory diagnosis,
and for what each is used.
Types of microscopy with
which you should be
familiar

Microscopy
plays a key
role in the
diagnosis of
infectious
disease.
When is microscopy useful?
• When organism’s morphology is rare
– Often parasites, where ovum, cyst, larval or
cellular appearance is characteristic
• When organism’s staining properties are rare
– Positive acid-fast stain = few agents
• Can be carried out on clinical specimen (direct
smear), or on cultured and/or isolated
microbes
• To help direct further tests
Types of microscopy used in diagnosis
• Light microscopy (brightfield)
– Wet mounts (usually larger cell
types), perhaps with phase contrast
– Stains such as Gram, acid-fast,
capsule, Giemsa
– Not valuable from sites w/normal
microbiota; isolation first
• Darkfield microscopy
(spirochetes)
Types of microscopy (con’t)
• Fluorescence microscopy
– Autofluorescence: few organisms
– Fluorescent stains:
• Chemical with direct affinity for a
microbial component
–Calcofluor white for fungi
–Auramine O for mycobacteria
• High sensitivity but lower specificity
than immunofluorescent methods
– Immunofluorescence
• Sensitive and specific
Fluorescence microscopy:
Immunofluorescence
• Fluorescent
molecule conjugated
to antibody
– Specific Ab = high
specificity
– Direct Fluorescent
Antibody (DFA)
– Indirect Fluorescent
Antibody (IFA)
LOs: Culture-based techniques
6. Describe the principles and methods used for the
cultivation of microbes.
1.Explain how each medium in the given table is
selective and/or differential.
2.Name the organisms these media select for
and/or differentiate and describe their resulting
appearance.
3.Describe the principles used for the cultivation of
obligate intracellular organisms including viruses.
Culture-Based Testing
• Requires viable organisms
• Requires ability to propagate
– Media
– Conditions
• Slow
• Usually followed up by further investigation to
ID what has grown
– Biochemical tests
– Typing (genotype, phage, etc.)
– Antimicrobial susceptibility testing
When is culture useful?
• Best for bacteria or fungi that grow readily
and are easily identified
• Usually necessary for antimicrobial
susceptibility testing
• Avoid culture when…
– Agent is extremely infectious
– Agent causes disease with no cure
Initial medium selection based on
specimen type
Specimen Type of Media Example of media
Sputum Media that supports growth of Blood agar (BAP)
most organisms Chocolate agar (CAP)
MacConkey agar (MAC)
Stool Media that supports growth of BAP
most organisms plus media MAC
selective for organisms that HE agar
commonly cause gastroenteritis EMB agar
Salmonella-Shigella agar
XLD agar
SMAC agar
Campy-BAP
Blood Media as suggested by Gram BAP
(if it flags stain Others depending on Gram
positive) stain results, e.g. MAC
See it for yourself –
markmicrobiology, DrKimmitt
• Stool
– https://www.youtube.com/watch?v=UtkFTIcA9pg

• Sputum
– https://www.youtube.com/watch?v=LuxlH5q6Pzg

• Blood
– https://www.youtube.com/watch?v=-d7pers6LTM
– https://www.youtube.com/watch?v=ZZoIZkna4vo

• Urine
– https://www.youtube.com/watch?v=T3rClVRCj6s
In your handout….

YOU MUST LEARN THESE DIFFERENTIAL
AND SELECTIVE MEDIA!!!!!!!!!
IS IT SELECTIVE, DIFFERENTIAL, OR BOTH?
HOW IS IT SELECTIVE?
WHAT DOES IT SELECT FOR/AGAINST?
HOW IS IT DIFFERENTIAL?
WHAT DO DIFFERENT ORGANISMS LOOK
LIKE ON IT?
Conditions for Growth
– Temperature
– Oxygen
• Strict aerobes
• Microaerophiles
• Facultative anaerobes
• Aerotolerant anaerobes
• Strict anaerobes
– Increased CO2
– Growth factors, nutrients
– pH
– Replication time (incubation time)
– Can get important clues from these…
Colonies grow…
Subculture to isolate colonies
http://www.wales.nhs.uk/sites3/page.cfm
?orgid=379&pid=13002

http://www.life.umd.edu/classroom/bsci424/pathoge
ndescriptions/StaphylococcusImages.htm

Copyright © 2007 Aerotech P&K
Identification of Organisms
• Important tools for identification of bacteria
grown in culture:
– Conditions for growth
– Colony morphology
– Cell morphology and arrangement
– Staining properties
– Biochemical properties
Biochemical Properties
• Bacteria have biochemical properties that can
be used to determine their identity
– Some make enzymes that can be detected
– Some use a particular biochemical process that
can be observed
LOs: Biochemical Tests
7. Explain the principles of the following
biochemical tests used in the identification of
bacteria isolated in culture: catalase, oxidase,
coagulase, urease, carbohydrate fermentation,
H2S detection, esculin production, thioglycollate,
bile solubility, hemolysis.
Perform tests on isolated colonies to
identify the organism

microbeonline.com
Biochemical Properties: Enzymatic activity

• Catalase test: looks for breakdown
of hydrogen peroxide to water and
oxygen (2H2O2  2H2O + O2)

• Oxidase test: detects presence of
cytochrome c oxidase
• An electron transport chain may have
one of two cytochromes in that position
that can’t oxidize the indicator
• Oxidase (+) = has cyt c oxidase, so can
use O2 in respiration
• Oxidase (-) = does not have cyt c oxidase,
so may have no ETC (and not use O2) or
may have alternate cytochrome oxidase
(and use O2+)
In your handout...

YOU MUST LEARN THESE
BIOCHEMICAL TESTS!!!!!!!!!
WHAT IS THE TEST LOOKING FOR?
WHAT DOES THE TEST HAVE IN IT?
HOW DOES A POSITIVE RESULT
OCCUR?
WHAT ORGANISMS DOES IT HELP
Urease Detects the presence of the Overnight
urease enzyme, which breaks
down amides (urea), producing
ammonia which will result in an
alkaline pH and a color change
to pink. Can be agar slant (left)

IDENTIFY?
or broth (right). Useful for
identifying Helicobacter,
Proteus, Ureaplasma, Nocardia,
and Cryptococcus.

Thioglycollate Thioglycolate removes oxygen. A tube of Overnight
semi-solid medium containing
thioglycolate is inoculated with the test
organism and incubated under normal
conditions, allowing oxygen to diffuse into
the medium from the top. This produces
an oxygen gradient in the tube. Obligate
aerobes will grow only at the top (1),
facultative anaerobes will grow throughout
but usually better at the top (2), aerotolerant anaerobes will be able to grow
throughout most of the tube (3), and strict anaerobes will grow near the
bottom of the tube (4). Where would a microaerophile grow?
Automation
• More rapid result, higher throughput, less tech time
• High initial costs
• Only do Gram, ox/cat, hemolysis, coag by hand
• Organism ID, antibiotic susceptibility testing
MALDI-TOF
• Matrix-assisted laser desorption/ionization-time of flight mass
spectrometry
• Measures exact masses of numerous proteins
• Produces a protein “fingerprint” for each organism
• Can identify cultured organisms quickly
Culture-Independent Diagnostic Tests
(CIDTs)
Detection of Infectious Agents

Direct microscopic examination (visualization)

Detection of a specific microbial product

Detection of specific antibodies against a
particular pathogen
LOs: Detection of other microbial
products

8. Explain how detection of microbial antigens and
nucleic acids can be used to diagnose an infectious
disease.
1.Explain how antibody agglutination, sandwich ELISA,
and chromatographic immunoassay techniques work
and when they can/should be used.
2.Explain how microbial nucleic acid detection methods
work, and when they are useful.
Detection of a specific microbial
antigens
• How do we detect antigens? With
antibodies!
– Agglutination
– Immunoassays
– Immunofluorescence
• We are detecting microbial antigens, not
patient immune responses to these
antigens!
When is detecting microbial
antigens useful?

• When antigen is produced in high enough
amounts to be detected
• When speed is crucial to prognosis
– Example: antigen detection assays on CSF
specimens for causes of meningitis
• Other confirmatory assays are usually
necessary!!!
Detection of Microbial Antigens
• Antibody agglutination
– Rapid (minutes), visual
– Works in treated patient
– Low sensitivity, specificity
– Qualitative
– On specimens or cultured material
Latex agglutination test
Detection of Microbial Antigens
• Sandwich Enzyme-Linked Immunosorbent Assay
– DIRECT ELISA = measures ANTIGEN (Ag)
– Targets 2 different epitopes on the Ag
• One is immobilized Ab, captures Ag from sample
• Second is enzyme-labeled Ab, detects captured Ag
(direct)
– Quantitative, relatively quick if no culture step
– On specimens or cultured material
Detection of Microbial Antigens
• Chromatographic Immunoassay
• Capillary action brings dye-conjugated Ag-Ab
complex to an immobilized capture Ab
– a sandwich ELISA done backwards
• aka Lateral Flow test
• Dipstick or strip formats

Thermo Fisher

Forms colored band or spot
• Rapid (minutes)
• Qualitative
• e.g. Rapid Strep test

NASA
Detection of Microbial Antigens

• Fluorescence microscopy described earlier
(direct or indirect), when the analyte is a
microbial product
– You are looking for microbial antigens in a tissue,
smear, bacterial culture, etc.
Detection of Nucleic Acids

• PCR, RT-PCR, real time PCR
• Amount of microbial NA can be quite small, so
need to amplify in some way to see signal -
SENSITIVE
• Based on binding of short pieces (fragments) of
DNA to complementary DNA sequences -
SPECIFIC
• Rapid (real time within hours), some direct on
specimen but most on cultured material
When is nucleic acid testing
useful?

• Difficult-to-cultivate organisms: obligate
intracellular organisms, unculturable, high risk
• Multiple possible causative agents: can screen
for more than one pathogen at a time
• Need for quantitation: good for tracking viral
loads to manage therapy
• Quick result: Often faster than biochemical ID
• Early result: Low pathogen numbers, no
waiting for immune response
Uses of PCR-based techniques
• Standard of diagnosis for
• HPV
• Chlamydia trachomatis & Neisseria gonorrhoeae
• Routine for quantification of viral loads of
• HIV
• HBV
• HCV
• Regularly used in diagnosis of
• HSV, CMV, EBV, VZV…
• Tick-borne infections
• Respiratory virus infections
Culture-Independent Diagnostic Tests
(CIDTs)
Detection of Infectious Agents

Direct microscopic examination (visualization)

Detection of a specific microbial product

Detection of specific antibodies against a
particular pathogen
LOs: Antibody detection in
Microbial Identification
9. Explain how infectious diseases are diagnosed
through the detection of pathogen-specific
antibodies from patient serum (continued in
Serological Techniques Workshop).
1.Recognize when and for what types of microbes
detecting antibodies is a useful tool.
2.Discuss the advantages and limitations of
serological techniques.
Antibody detection in Microbial ID
• Indirect way of inferring presence of organism or microbial
product, by looking for pathogen-specific host antibodies
• Used in particular cases:
– When pathogen is dangerous, difficult to grow, inaccessible or in low
numbers
– To track course of illness (acute to convalescent)
– To determine immune status and disease status
• Commonly used for diagnosis and management of numerous
infections:
– Viruses (CMV, VZV, Hepatitis Viruses, Influenza and more)
– Intracellular or difficult to grow bacteria (Treponema pallidum,
Leptospira, Chlamydia, Rickettsia, and many more)
– Fungi (Histoplasma, Coccidioides, Blastomyces, Candida)
Host Antibody Response

• Type of
antibodies seen
and change in
titer over time
can give you idea
of
– Disease stage
– Treatment
success (or
failure)
Titer
• Highest dilution of serum able to agglutinate particular antigen
– Quantitative: Compare to standards to obtain a concentration
– Semi-quantitative: reported as ratio based on dilution (1:8, 1:16,
1:32…), or its inverse (8, 16, 32)
– Qualitative: positive/negative, reactive/non-reactive
• Mix serial dilutions of serum with constant amount of antigen
bound to particle/cell, look for agglutination – cross-linked particles
won’t settle to bottom
• Higher dilution means more specific antibody present initially
Detection of specific antibodies against a
particular pathogen
Advantages Disadvantages
• Usually very sensitive and • IgM antibodies can only
specific be detected early in
• Can tell if protective response infection (7-10 days)
exists • IgG antibodies may not
• IgM antibodies indicate acute appear for 2-4 weeks, so
or active infection diagnosis may be
• Compare acute and retrospective
convalescent sera to show
• May not know if infection
evidence of infection
past or present
– usually 4-fold rise in titer,
e.g. from 1:4 to 1:16
READ THE MATERIALS
ASSOCIATED WITH THE
SEROLOGICAL TECHNIQUES
WORKSHOP BEFORE THE
WORKSHOP!!!
Indirect ELISA
• INDIRECT ELISA = looking for AB
• Specific Ag used to capture specific Abs from
patient serum
• Detected with labeled anti-human Ab (indirect)
• Quantitative if compared to standard curve
Western Immunoblot
• Microbial Ag separated and blotted to
membrane
– Commercially-prepared, e.g. HIV proteins
• Membrane incubated with patient serum
• Membrane then incubated with labeled
anti-human Ab
– Patient Ab bound to Ag will be marked
with label
• Bands containing pathogen proteins that
have provoked a response will be labelled
What test uses the same process?
LAST LO…
10. Differentiate microbial pathogens based on
key characteristics. Use the TESTABLE BACTERIA
list for guidance.
TESTABLE BACTERIA:
FOR ALL in the flowchart, know at least the Gram stain and morphology.
Further provided detail for:
Mini 1: Staphylococcus spp., Streptococcus spp., Enterococcus
Bacillus, Clostridium, Listeria, Corynebacterium, Nocardia, Mycobacterium
Citrobacter, Enterobacter, Escherichia, Klebsiella
Shigella, Yersinia, Proteus, Salmonella, Serratia
Treponema, Borrelia, Leptospira, Mycoplasma
Mini 2: the above PLUS
Lactobacillus, Actinomyces
Porphyromonas, Prevotella, Bacteroides
Neisseria spp., Acinetobacter, Pseudomonas
Campylobacter, Helicobacter, Vibrio
Legionella, Rickettsia, Ehrlichia, Coxiella, Chlamydia, Chlamydophila
Later: all the rest and more…and more information about many
IN ALL CASES, any organisms discussed by other professors
Study strategy
• On index cards, write the organism name one
one side, important characteristics on other
side (leave lots of space, you’ll have more to
add!!!)
• Use with or without a flowchart outline to
classify and differentiate genera
– Gram result, morphology, biochemical reactions,
symptoms, diseases, syndromes etc. etc. etc.
You need to know how to interpret
lab data
• Sample culture report

Smith, John 0767052

Blood culture:

Gram-negative cocci, pending identification
• Catalase: breaks down hydrogen
peroxide to water and oxygen
2H2O2  2H2O + O2
Gram positive cocci
Staphylococcus Streptococcus Enterococcus

• Grape-like • Pairs or chains • Pairs or chains
clusters
Staphylococcus spp.
• Gram positive cocci in clusters, catalase (+)
• S. aureus is most important pathogen
– Coagulase (+)
– β-hemolytic Mannitol Salt Agar
– Yellow colonies (often)
– Salt tolerant (haloduric)
– Produces acid from mannitol
Staphylococcus spp.
• Coagulase-negative staphylococci (CoNS)
– Most are normal flora and cause disease less
often, and less severely, than S. aureus
– S. saprophyticus and S. epidermidis are important
pathogens
• These can be differentiated based on susceptibility to
novobiocin

S. epidermidis S. saprophyticus
Streptococcus spp.
• Gram-positive cocci in pairs or chains
• Important characteristics
– Catalase-negative
– Hemolysis patterns help in identification of
species
– Lancefield antibodies to cell wall
carbohydrates are used to classify streptococci
into serogroups A-U
S. pneumoniae S. pyogenes Enterococcus
Viridans streptococci S. agalactiae Some other strep
Streptococcus spp.
• Some members of the genus are part of normal
human flora while others are important human
pathogens
α-hemolytic streptococci
S. pneumoniae

S. mitis
β-hemolytic streptococci

(pyrolidonyl
arylamidase)
Non-hemolytic streptococci:
Enterococcus

• Members of streptococcal family
• Share growth characteristics with streptococci
• Lancefield Group D antigen positive
• Resistant to bile salts
– Will grow on bile esculin agar
– Some species will hydrolyze esculin
(black precipitate on bile esculin agar)
Enterococcus
• Part of normal GI flora and seldom cause disease
in normal, healthy individuals
• E. faecium and E. faecalis most important species,
require further biochemical testing to
differentiate
• Frequent cause of nosocomial infections
• Highly resistant to environmental and chemical
agents
• Antibiotic resistance is a major problem
– these organisms can be resistant to multiple anti-microbials
(either intrinsically or through acquired resistance plasmids)
Gram-negative
bacilli Enterobacteriaceae
Facultative
anaerobes • Normal GI flora
• Ferment glucose
Oxidase (-) • Grow rapidly on non-selective media
• Cytochrome oxidase (-)
• Lactose utilization is the best next step
in identifying Gram (-) oxidase (-)
facultative rods
• Multiple tests required to get down to
genus (indole, citrate, MR-VP, lysine
decarboxylase, motility, urease, H2S)
ENTEROBACTERIACEAE

Non-Lactose-
Lactose-fermenting
Fermenting

Citrobacter Shigella
Escherichia Yersina
Enterobacter Proteus
Klebsiella Salmonella

Serratia: can be Lac (+) or (-), but colonies produce red pigment
Many other genera but of lesser clinical importance
Lactose-fermenting
Enterobacteriaceae
Citrobacter
• Less common; CNS infections in immunocompromised
Enterobacter
• Less common; lower resp tract, bloodstream
Escherichia (coli)
• Common, many types of disease
• Disease caused is often strain-dependent
• Often encapsulated, motile
Klebsiella
• Common, many types of disease
• Encapsulated, often highly mucoid
• Non-motile, produces urease
Other tests – H2S production
• Certain bacteria produce H2S from provided
sulfates as a byproduct of energy production
• Combines with iron provided in medium to
make FeS (↓)
• e.g. Triple Sugar Iron Agar

No H2S production H2S production
Other tests – Hektoen Enteric Agar
• Selective and differential for isolation E. coli
of enteric pathogens
– Bile salts and indicator dyes inhibit
the growth of Gram-positive
organisms
• Sugars: lactose, sucrose, and salicin
– fermenters = yellow-pink, orange
– non-fermenters = green or
transparent Shigella
• Organisms that produce H2S will form Salmonella
a black precipitate on the colony
• Differentiation of Salmonella and
Shigella
Non-lactose
fermenting
Enterobacteriaceae Shigella
•Non-motile
Non-H2S - •4 species, limited to colon
producing
Yersinia
•Short, pleomorphic Gram-
negative rod (coccobacillus)
•Bipolar staining (“Safety-pin”)
•Growth in 48 h
•Y. enterocolitica (enterocolitis)
•Urease (+)
•Motile at 25°C, not higher
•Can grow slowly at 4°C
•Y. pestis (plague)
•Urease (-)
Wright-Giemsa stain (don’t be •Non-motile
confused because the
Non-lactose
fermenting
Enterobacteriaceae

H2S - Proteus
producing •Swarming motility
•Urease-positive www.microbiologyatlas.kvl.dk

•Cause of UTI, kidney stones

Salmonella
•Motile
•Important cause of gastroenteritis,
systemic infection, and enteric fever

www.textbookofbacteriology.net
Gram-negative bacilli
Straight rods

Facultative Anaerobes
Oxidase (-)
Enterobacteriaceae
• Ferment glucose
• Normal GI flora
• Multiple tests required to get down to genus (indole, citrate, MR-
VP, lysine decarboxylase, motility, urease, H2S)
• Serratia can be lac(+/-), often produces red pigment

Lactose-fermenting (CEEK) Non-lactose fermenting (ShYPS)

Citrobacter Escherichia Non-H2S -producing H2S -producing
•Motile •Motile Shigella Proteus
•Urease (+/-) •Urease (-) •Non-motile •Swarming motility
Enterobacter Klebsiella •Urease (-) •Urease (+)
•Motile •Often highly mucoid Yersinia Salmonella
•Urease (+/-) •Non-motile •Bipolar staining •Motile
•Urease (+) •Urease (+/-) •Urease (-)
•Motility (+/-)
Summary
• Specimen collection is very important
• Lab identification is a process—MD and
microbiologist work together
– Symptoms, direct microscopy suggest further
tests
– Culture-based or non-culture-based tests
– Serologic testing
• When applied and interpreted correctly,
allows correct diagnosis and treatment