Fundamentals 2 Lab Diagnosis and Bacterial ID Dr.

Drummelsmith

Laboratory Diagnosis and Bacterial Identification

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.

References: Murray Medical Microbiology, 8th edition, Ch. 4, 5, 6 (Vital Source)

Sherris Medical Microbiology, 7th edition, Ch. 4 (Access Medicine)

Learning activity: In this guide

Practice Questions: Canvas quiz

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

1) Effective diagnosis and treatment of infectious diseases is dependent on an accurate and complete
clinical history, interpretation of signs and symptoms, physical examination, and often the
results of appropriate laboratory investigations.
a) Sometimes, an infection can be diagnosed clinically, and the physician does not need any
information from the laboratory to appropriately manage the patient.
i) There are some pathogens that produce clinical signs and symptoms that are
pathognomonic, or characteristic or indicative of a disease. For example,
pseudomembrane on tonsils, pharynx and nasal cavity in diphtheria.
ii) With other pathogens, there are no laboratory tests available (either they don’t exist or they
are too expensive) so diagnosis is largely based on the clinical signs and symptoms. For
example, the diagnosis of trachoma is largely clinical, as it affects mainly impoverished
children and sophisticated laboratory tests are simply unavailable in these areas.
b) The clinical microbiology laboratory plays a central role in identifying pathogens,
products of pathogens, and the host response. All laboratory studies should be directed
by the results of the H&P and then evaluated taking into account the sensitivity and specificity
of the tests.
c) Pathogen identification is often key to the successful treatment of infectious disease, as
treatment is becoming more and more tailored to specific etiologies
i) Narrow-spectrum antibiotics
ii) Drug-resistant organisms
d) Goals of diagnostic microbiology include:
i) Identifying the presence or absence of microbes in a clinical sample
ii) Characterizing microbes for phenotypes including toxin production and antibiotic
susceptibility

2) The first step to successful laboratory diagnosis is

correct collection and handling of the clinical
specimen.
a) The sample must come from a site likely to
contain the analyte or causative agent of the
suspected illness.
b) The sample must be taken during a phase of
the disease when the agent or analyte will be
present.
c) Antimicrobial treatment can hinder diagnostic
techniques requiring growth or enumeration, so
ideally samples should be taken before
treatment has begun.
d) Enough material should be collected to allow
repeated or additional (reflex) testing, as some
tests may require follow-up depending upon
initial results.
e) Contamination must be considered.
i) Many clinical specimens are sampled from
non-sterile sites. It is important to be able to
distinguish non-pathogenic microbes from
pathogens or from potential opportunistic
infections. A good knowledge of the normal
microbiota (flora) is essential for correct
interpretation of diagnostic results.
ii) Clinical specimens from normally sterile sites may have to be obtained through non-sterile
sites. For example, urine samples must pass through the urethra and skin. It is important
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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

that appropriate collection techniques be followed to minimize such contaminants (surface

cleansing, midstream urine collection).
iii) Equipment used for collection (containers, swabs, etc.) should be sterile and used carefully
to prevent contamination via handling.
f) Media and containers designed for the sample type and suspected pathogen should be used to
preserve the integrity of the sample and the viability of microbes within it.
g) Specimens must be labeled as to the source of the sample, patient information
(name/ID#/room#), along with time and date of collection.
h) Because diagnostic microbiology requires a significant degree of interpretation on the part of
the microbiologist, as much clinical information as possible, including likely causative agents,
should be provided.
i) Be sure to ask for all needed tests (for example, non-standard antibiotic susceptibility profiles in
the case of a patient with known treatment failure).
j) Transport to the lab should be carried out as quickly as possible.

3) Basic Flow of Laboratory Diagnosis

a) Direct examination of the patient specimen
b) Growth and cultivation (i.e., culture) of the causative agent(s)
c) Analysis of the cultured agent(s) to establish identification and other characteristics
The diagram/algorithm below outlines what happens once the specimen gets to the laboratory:

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

d) Interpretation of results is key to correct diagnosis and treatment. It is important to

understand the principles underlying different tests, not only to order the correct ones but also
to be clear on what the results mean. Few tests (if any) will be 100% correct, and the incidence
of disease in a given population will influence how often incorrect results are obtained. Tests
are evaluated by the following parameters:
i) Sensitivity: probability that the test will correctly identify a positive sample (TP)
ii) Specificity: probability that the test will correctly identify a negative sample (TN)
iii) Positive predictive value (PPV): proportion of true positives among test positives
iv) Negative predictive value (NPV): proportion of true negatives among test negatives.

e) There are several types of diagnostic tests that can be carried out using material already
present within the clinical specimen. These techniques do not require growth or isolation of
microbes. They are generally more rapid than culture-based methods and cost varies.

4) Direct Microscopic Examination (Visualization). Clinical specimens are examined directly by

microscopy to assess the quality of the specimen, to determine if visible microbes are present or
absent in the specimen, and to help identify those organisms present. Microscopy is also used to
help identify isolated organisms after culture.
a) Light microscopy is the most common type of microscopy. Specimens can be observed as
wet preparations or by fixing and staining a slide. Limit of resolution is ~0.2 µm. Good for
studying microbes larger than typical viruses and internal structures of larger microbes.
i) Wet mounts allow microbes to be seen while alive, which can allow detection of motility.
They may be used for several types of samples:
(1) Fluids – urine, cerebrospinal fluid, or feces
(2) Larger cells such as parasite cysts or eggs, protozoa, and some fungi
(3) Fungi from skin samples suspended in a potassium hydroxide (KOH) solution which
dissolves skin cells to allow better visualization
ii) Stained preparations involve drying and fixing the specimen to a glass slide and
application of particular chemicals. Common staining techniques include:
(1) Gram stain
(2) Acid-fast stains (e.g. Kinyoun, Ziehl-Neelsen)
(3) Capsule stains, such as India ink or quellung reaction
(4) Spore stains
(5) Giemsa or Giemsa-Wright stains
(6) Silver stains
b) Darkfield microscopy: The light microscope is adapted by modifying the condenser to
prevent transmitted light from directly illuminating the unstained specimen. Light passes

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

around (reflected) rather than passing through the organism; the specimen appears brightly
illuminated against a black background which can make extremely thin cells, such as
spirochetes, easier to see.
c) Phase contrast microscopy: Also facilitates visualization of unstained samples, but by
exploiting differences in light refraction rather than reflection to give more of a 3-dimensional
appearance and better visualization of internal structures.
d) Fluorescence microscopy: This method takes advantage of fluorescent compounds, which
are molecules that absorb energy at one wavelength and emit some of that energy at
a longer wavelength. When the emitted wavelength is in the visible range, cells stained with
these compounds appear colored. There are two major types of fluorescent visualization:
i) Fluorescent staining, using compounds such as calcofluor white (fungi) or auramine
O-rhodamine (mycobacteria), in which the fluorescent compound interacts directly with
the microbe. These are often less specific than targeted dyes (discussed below).
ii) Targeted fluorophores or fluorescent dyes, which are most often conjugated to
antibody (immunofluorescence microscopy).
(1) Direct: Antibody specifically recognizing a microbial antigen or other structure is
conjugated to a fluorophore.
(2) Indirect: Antibody specifically recognizing a microbial antigen or other structure is
tagged with an Fc-specific fluorophore-conjugated secondary antibody.
e) Electron microscopy: The wavelength of electrons is much smaller than that of photons,
allowing high resolution and visualization of smaller particles (such as viruses and organelles).
Scanning EM (SEM) gives an image of the surface of a specimen, whereas transmission EM
(TEM) uses stained thin sections to provide images of internal structures.

5) Cultivation of causative agents, and their subsequent identification, are still the mainstay of
many diagnostic procedures. In general, these require a sample containing viable organisms
(which is why collection and transport are so important), as well as growth medium and conditions
that allow propagation of the species of interest.
a) Often, bacteria and fungi can be grown in vitro with the right combination of nutrients and
environmental conditions. Nutrients are often mixed with a semi-solid support, known as agar,
and usually poured into plates, which allows isolation of colonies derived from individual
organisms. For many microbes, a rich general medium is sufficient for growth. However,
fastidious organisms require specific growth factors (that may or may not be known) and,
therefore, specialized media for growth.
i) The composition of complex media are undefined, in that they may contain infusions
made from organs (e.g. brain-heart infusion), yeast extract, blood, and/or digested proteins.
Defined media contain known quantities of several purified components, and can range
from simple (e.g., glucose plus a few salts) to very complex (e.g., sugars, salts, lipids, and
numerous growth factors).
ii) Media may contain selective agents, which inhibit certain classes of organisms from
growing. These are useful when samples come from non-sterile sites and the commensal
microbiota may be numerous or rapidly outgrow the suspected pathogen. They also aid
identification, as fewer species can grow on them. Antibiotics may be used to inhibit growth
of particular bacteria in fungal/yeast cultures or in culturing specimens commonly
contaminated with normal microbiota.
iii) Media may also contain differential agents, which allow species to be distinguished from
each other based on how they react with the differential agent.
iv) Selective and differential agents are commonly combined into a single plate or tube.
b) The nature of the specimen greatly informs the method of culture chosen.
i) While most specimen types are usually inoculated onto semi-solid media, blood is usually
inoculated into liquid media (in sealed bottles, called “blood cultures”), and incubated both

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aerobically and anaerobically. Other high-volume liquid specimens may also be inoculated
into liquid media in some cases.
ii) Specimen type will also determine which specific media are used. For most specimen types,
the laboratory selects media that promote the isolation of pathogenic organisms that are
frequently isolated from that anatomic site. For example:
(1) Sputum: Respiratory infections are commonly caused by both Gram-negative and Gram-
positive organisms (and poorly Gram-staining organisms). As such, a sputum specimen
would be inoculated onto media that supports the growth of most organisms, both
Gram-positive and Gram-negative (e.g. blood agar).
(2) Stool: Most pathogens causing gastroenteritis are Gram-negative, so stool is typically
inoculated onto media that restricts the growth of Gram-positive organisms in an effort
to better isolate the Gram-negative organisms (e.g., blood agar + MacConkey agar).
Media that isolate and/or differentiate particular pathogens are also included in the
media panel (for example, Sorbitol MacConkey agar for the isolation of E. coli O157:H7,
TCBS agar to better isolate Vibrio sp.). In order to be thorough, the stool specimen is
also inoculated onto a general purpose medium, such as blood agar, that supports both
Gram-positive and Gram-negative organisms.
c) Important media used for the isolation, selection, and/or differentiation of bacteria
(there are others, these are a good start):

Agar Description Image

Blood agar General purpose and differential medium used to isolate a variety
(BAP) of microorganisms. Usually made with sheep blood. Provides many
growth factors for fastidious organisms. Pattern of hemolysis
(alpha-α, beta-β, gamma-ɣ) can aid in identification.

Chocolate Contains blood that has been heated to lyse red blood cells (turns
agar the blood brown) and release intracellular nutrients (e.g. NAD,
(CAP) hemin). Useful for organisms with difficult nutritional
requirements that cannot lyse the cells themselves, such as
Thayer- Haemophilus and Neisseria. A selective version of this medium
Martin agar called Thayer-Martin for isolation of Neisseria gonorrhoeae has
antibiotics added.
Mannitol- High salt concentration inhibits all but osmotolerant organisms.
salt agar Fermentation of mannitol results in acid production, causing a
(MSA) drop in pH and turning pH indicator from red to yellow. Useful in
selecting for staphylococci (haloduric = osmotolerant = salt-
tolerant), and differentiating S. aureus (ferments mannitol) from
coagulase-negative staph (do not ferment mannitol).
MacConkey Contains bile salts and crystal violet to inhibit growth of Gram-
agar positive organisms and some fastidious Gram-negative bacteria
(MAC) (e.g., Hemophilus and Neisseria), making it selective for some
Gram-negative organisms. Contains lactose as the sole
Sorbitol carbohydrate, and neutral red as the pH indicator. If the organism
MacConkey ferments lactose, acid production lowers pH and colonies appear
agar (SMAC) pink (e.g., Escherichia coli). If the organism is a non-lactose
fermenter, colonies will appear colorless or beige (e.g.,
Salmonella). Can swap out the sugar for another (e.g. Sorbitol
MacConkey for identification of E. coli O157:H7)

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

Eosin Similar in principle to MacConkey agar, except different indicators

Methylene (eosin and methylene blue) that inhibit Gram-positive organisms
Blue agar and change color with pH. Lactose-fermenters appear as blue-
(EMB) black colonies, while non-fermenters appear clear. Organisms that
are strong lactose fermenters, such as E. coli, can have a shiny
green metallic sheen.
Bile esculin Bile salts inhibit the growth of Gram-positive organisms and most
agar streptococci except Group D Streptococcus and Enterococcus.
(BEA) Esculin can be hydrolyzed by Enterococcus and Group D
Streptococcus to esculitin, which interacts with iron to form a
black precipitate of ferrous sulfate.
Can be plate or tube.
Hektoen Bile salts and indicator dyes inhibit the growth of Gram-positive
enteric agar organisms. Fermenters (sugars – lactose, sucrose, and salicin)
(HE) appear yellow-orange, and non-fermenters appear green or
transparent. Organisms that produce hydrogen sulfide (H2S) from
sulfate (e.g., Salmonella but NOT Shigella) will form a black
precipitate resulting from the interaction with ferric ammonium
citrate in the medium.
Cetrimide For the isolation of Pseudomonas aeruginosa. Cetrimide is a
agar detergent that inhibits most bacteria except pseudomonads.
Contains magnesium and potassium which promote the
production of the pigments pyocyanin (blue-green) and
fluorescein (yellow-green) produced by some Pseudomonas spp,
such as P. aeruginosa.
Tellurite For isolation of Corynebacterium diphtheriae. Potassium tellurite
agar inhibits Gram-negative organisms and most upper respiratory flora
(Cystine- except Corynebacterium spp. C. diphtheriae colonies appear gray
tellurite or black (due to tellurite reductase activity) with a brown halo
agar) (cysteinase activity) around the colonies.
Triple Sugar Contains lactose, sucrose, a small amount of glucose, protein
Iron (TSI) digest, sodium thiosulfate, ferrous iron, and a pH indicator.
agar Location of yellow color change (from the original pink) indicates:
no yellow, no sugar utilization (A); entire tube, fermentation of
multiple sugars (B); butt only, glucose fermentation only (aerobic
use of peptides produces ammonia, neutralizing the top; C and D).
H2S production produces a black precipitate (C).

Chromo- Usually designed to differentiate organisms of interest though a

genic agars change in colony color. These have been developed to identify
specific species, such as Listeria monocytogenes or Staphylococcus
aureus (SA), or to identify specific phenotypes such as methicillin-
resistant SA (MRSA) versus methicillin-sensitive SA (MSSA). They
are specific and quicker as growth can be easily seen.

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

d) As with bacterial cultures, specimen type influences the type of media used for fungal culture.
i) Media
(1) Sabouraud dextrose agar is a good general-purpose agar for the growth of all fungi.
(a) Different variations of Sabouraud agar are used – these variations include antibiotics
to some fungi and bacteria or the addition of additional nutrients to enhance the
growth of some fungi.
(2) Other selective or differential agars exist to select for or differentiate various fungi.
ii) Incubation: Cultures are incubated for much longer than bacterial cultures (weeks vs. days).
iii) Identification: Colonial and microscopic morphology, biochemical tests (primarily done with
yeasts), or by immunologic tests, molecular probes, or DNA sequencing.
e) Viruses and other obligate intracellular organisms have an obligatory requirement
to grow and multiply within eukaryotic cells. As such, they cannot be cultivated on
independently semi-solid media; in order to be cultivated in culture, they require a host cell.
These organisms can be propagated inside tissue or cell lines.
i) Specimen type plays a slightly different role. Rather than influencing which media are
inoculated, specimen type influences the type of tissue or cell line that is
inoculated. Certain viruses infect specific anatomical sites; therefore, the cell lines that are
chosen are best suited to support the growth of the viruses that are the most likely to be
causing the infection.
ii) Tube cell cultures: When growing in culture, cell lines will often adhere to a surface. For cell
culture for laboratory diagnosis of infection, specimen is added to a glass tube. The walls of
the glass tubes are lined with tissue or cell lines.
iii) Different viruses require different cell types to support their growth. For example,
herpes simplex virus grows well on human diploid fibroblast cells or on human epidermoid
carcinoma lines. Chlamydia grows well in HeLa or HEp-2 cell lines.
iv) Identification:
(1) The classical method for identification of a virus is by observing the cytopathic effects
(CPE) or changes the virus has on the cell line. Different viruses have different patterns
of CPE, and these patterns can be used to identify the virus growing in culture. The tube
cells are observed under low-magnification for evidence of CPE.
(2) Alternatively, immunologic methods (fluorescent antibody staining) can be used to
identify the virus.
(3) Some microbes cannot be propagated on artificial media. They are either obligate
intracellular organisms (e.g., viruses, rickettsia, and chlamydia), or we have not
figured out their nutritional or environmental requirements. Some of these may be
grown in tissue culture cells; others must be propagated in animals. Non-culture-
based identification methods are used if they exist.
6) Important tools for the identification of bacteria grown in culture take advantage of
particular bacterial properties, including staining properties, cell morphology, conditions for growth,
and biochemical properties. Microbiologists have tests that analyze these properties and link the
property with the organism’s identity.
a) Some microscopic and non-culture-based techniques can be used equally well on cells derived
from specimens or culture, but many identification methods are dependent on the purity and
cell numbers obtained after isolation and culture. Once the initial sample has been grown and
individual organisms isolated and grown again, they can be used for further identification tests.
Note that some of these tests may also be built into a differential medium (above).
i) Growth characteristics: Sometimes, the conditions required to grow an organism
(aerotolerance, required cofactors) will provide strong clues as to the genus or species and
will help direct the choice of further assays. Other tests also take advantage of
(1) Ability to use/requirement for a particular nutrient, often a sugar, but could be
something else (e.g. an alternate carbon source).

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

(a) Ability to ferment a particular sugar is a common example of this, such as

mannitol or lactose, with or without the production of gaseous byproducts.
(2) Ability to grow under particular conditions, such as different oxygen tensions as
seen in the thioglycollate test.
ii) Biochemical assays: These tests are used to identify the presence of specific enzymes or
pathways. Some of these could also be classified as testing growth characteristics, but
generally depend on the presence of a single enzyme rather than an entire pathway. Results
may be obtained rapidly (within a few minutes) or may require overnight growth to
interpret. Examples are:
(1) Ability to catalyze a particular reaction, such as the catalase test (enzyme that
converts peroxide to oxygen and water), or lecithinase test (enzyme that degrades
lethicin). The esculin test and H2S production tests have been described above with Bile-
Esculin Agar and Hektoen Enteric Agar.
(2) Certain bacteria alter the appearance of a BAP in
characteristic ways, referred to as hemolysis. The
presence of and type of hemolysis gives clues to the
organism’s identity. Types of hemolysis:
(a) -hemolysis: partial lysis of the RBCs; organism
produces hydrogen peroxide, resulting in the oxidation
of hemoglobin to green methemglobinperoxide
(b) -hemolysis: complete clearing of the RBCs around
the bacterial colony; organism produces hemolysin
enzyme which is responsible for the lysis of the RBCs
(c) -hemolysis or non-hemolysis: bacteria have no
effect on the RBCS
iii) Antibiotic susceptibility can be useful in identification, and is commonly done to help
with treatment, but will be covered in another lecture.

Important biochemical tests (not including esculin and H2S production, described above)
Test Description Test Time
Catalase Detects presence of catalase Rapid –
enzyme, which converts hydrogen under a
peroxide to water and oxygen minute
(2H2O2  2H2O + O2); anaerobes
are less likely to produce catalase
(but several do!). Most helpful in
differentiating Gram-positive
bacteria.
Coagulase Detects enzymes which convert fibrinogen to fibrin; Staphylococcus aureus Slide – a
and Yersinia pestis are positive. Two types of coagulase and coagulase tests few
exist. It either is positive, then the organism is considered coagulase (+). minutes
 Free coagulase: secreted from bacterial cell. Identified with tube Tube –
coagulase test. Secreted bacterial coagulase reacts with prothrombin in overnight
the liquid test tube medium to form staphylothrombin, which catalyzes the
conversion of fibrinogen within the medium to insoluble fibrin, causing the
medium to gel.
 Bound coagulase (clumping factor): attached to bacterial cell wall. Tested
with slide coagulase test or coagulase agglutination test. Enzymatically
converts fibrinogen (attached to latex beads) to insoluble fibrin, causing
bacterial cells and beads to clump.

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

Fermentation pH indicators in the media reflect the Overnight

production of acid from metabolism of a
sugar in the medium; fermentation
patterns help with identification. Note
that several differential media use this
type of test, but that these do not
differentiate homo- (no gas production)
from heterofermentation (gas production,
caught in little tube).
Acid may also be produced oxidatively,
rather than fermentatively.
Oxidase Detects the presence of “cytochrome c oxidase”, a Rapid –
component of some electron transport chains, under a
which will turn a particular indicator purple. Some minute
bacteria with an ETC have a different molecule
replacing cytochrome c oxidase, and will test
negative. Helps to differentiate groups of Gram-
negative bacteria.
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)
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?

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7) Detection of microbial products can be used to infer the presence of a pathogen without
actually isolating it.
a) Chemical analysis for metabolites. Analytical chemistry techniques (such as gas
chromatography, mass spectrometry, etc.) may be used to identify small organic molecules that
may be produced by specific microbes. Costs and expertise associated with necessary
instrumentation often limit the use of these techniques.
b) Speciation by mass spectrometry. This method uses very precise measurements of ions
(usually proteins) to speciate an isolate. Expensive equipment, so not widespread, but is robust
and highly accurate.
c) Detection of antigens expressed or secreted by microbes (surface polysaccharides or
proteins, secreted proteins such as some exotoxins). DIRECT assays determine presence of
ANTIGENS in a clinical or cultured sample.
i) Direct antibody agglutination/detection of specific
antigens: Inert latex beads conjugated to specific antibodies (to
form a multivalent structure) are mixed with a specimen 
cross-linking with the antigen in the specimen occurs  results in a
visible clumping reaction. IgM antibodies will work much the
same way—the main thing is that the reacting antibody particle and
the molecule of interest can participate in more than one
interaction. Results can be obtained within minutes, but may be
prone to non-specific binding and low sensitivity (FP); usually
qualitative assays. This is called a latex agglutination test.
ii) Immunoassays:
(1) The sandwich enzyme-linked immunosorbent assay
(direct ELISA) and enzyme immunoassay (EIA) have become a mainstay of the
diagnostic lab for many things, not just microbiology. The use of an enzymatic detection
mechanism or fluorophore can amplify the signal, providing high levels of sensitivity.
The amount of signal is proportional to the amount of detection molecule present, which
is proportional to the amount of analyte. Comparison to a standard curve can provide a
quantitative result. The sandwich technique targets two different epitopes on the
analyte, one to capture and one to measure. Often, a polyclonal antibody is used for
capture and a monoclonal for detection to increase specificity.

(2) Chromatographic immunoassay (CIA): Available in dipstick or strip formats,

where capillary action brings a dye-conjugated antibody-
antigen complex to a second, immobilized capture
antibody. Concentration of the dye at the capture zone
leads to a visible spot or band.
iii) Biological assays: Time-consuming, specialized tests
performed only in specialty laboratories
(1) Injection of patient’s serum into mice (Clostridium
botulinum toxin)
(2) Enterotoxin detection by tissue culture (Clostridium difficile toxin) or animal model use

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d) Nucleic acid-based tests allow the causative agent to be identified based on detection of
specific nucleic acid signatures via hybridization to complementary nucleic acid sequences.
Many of these tests use nucleic acid amplification to specifically increase the amount of
material present, a technique known as the polymerase chain reaction (PCR), but some
amplify the signal instead (branched DNA assays).
i) Regular PCR: Amplifies a
particular DNA sequence. When
each cycle of the process is
repeated, this results in a doubling
of the amount of that DNA
fragment until enough is produced
to be visualized.
ii) Modifications of PCR
(1) Reverse-transcriptase PCR
(RT-PCR): Amplification starts
from RNA. The RT enzyme
converts the RNA into
complementary DNA
(cDNA), then regular PCR is
used to amplify the cDNA until
there is enough to be
visualized. It is particularly
useful for the detection of RNA
viruses in patient specimens
(2) Real-time PCR: Amplification
starts from DNA or cDNA. Similar to PCR except that it incorporates the visualization
of the product via fluorescence as amplification occurs (i.e., in “real time).
Primary benefits are speed and increased specificity due to the use of a sequence-
specific probe in addition to primers.
(3) Real-time RT-PCR: Although this method uses the same principle as real-time PCR, it
starts with a reverse transcription step.
iii) Branched DNA assays: In this
assay, the nucleic acid amplification
step is skipped. It amplifies the
signal, not the nucleic acid itself.
Instead, a single-stranded DNA probe
is generated that incorporates a
portion that is complementary to the
targeted sequence and a portion that
will bind an engineered branched
DNA structure. This branched
structure has been conjugated to
numerous enzymes which will create
a signal when mixed with a substrate.
Therefore, a single nucleic acid signal
will produce a highly-amplified signal,
and the amount of signal is
proportional to the number of copies
of the specific nucleic acid target
present in the sample.

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

8) Serologic tests are designed to diagnose an infectious disease by detecting the host’s response
against the pathogen in the form of antibodies. These techniques do not rely on the
identification of microbes or microbial products, but on the identification of the body’s response to
the organism. These can also provide information as to the progress of the infection. Here are just
a few; you will see more in the Serological Techniques workshop.
a) IgM indicates an early, active infection, whereas an IgG response takes several weeks
to occur. Detection of IgG does not mean that an infection is present, just that the patient was
exposed previously. Changes in titer (the measure of antibody present) can show disease
progression or treatment success.
b) Advantages:
i) Usually very sensitive
ii) Detection of IgM antibodies are indicative of acute or active infection
iii) Comparison of acute and convalescent sera can demonstrate evidence of infection (usually
4-fold rise in titer is the criteria for evidence of infection)
c) Disadvantages:
i) IgM antibodies can only be detected early in infection (7-10 days)
ii) IgG antibodies may not appear for 2-4 weeks, so the data, and potentially the diagnosis,
may be retrospective
d) Several serologic techniques used to detect the presence/absence (qualitative) and titre
(quantitative) of specific antibodies.
i) Measurement of antibodies (indirect ELISA, indirect fluorescent antibody (IFA))

(1) In this technique, test kits contain antigen from the

organism of interest. Patient serum is applied and
wells/slides are washed. If antigen-specific antibodies
are present they will bind and will not wash off.
Then, a human antibody-specific second antibody is
applied. In ELISA, this is enzyme-linked; in IFA, a
fluorophore is linked. If patient antibodies have
bound, this second antibody will bind them and will
remain through a wash step. The label then permits
detection, via measurement of either the product of
an enzymatic reaction, or of fluorescence.
(a) Advantages: May be the only method for
diagnosis of many organisms, particularly
intracellular pathogens.
(b) Disadvantages: Subject to interpretation by
laboratory technologists, who must be highly
trained. Also there are issues with cross-
reactivity.
ii) Complement Fixation
(1) This is an old but useful method for detection of antibodies against specific pathogens.
It exploits interactions of antibody with complement.

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

(a) Examples: These tests are still heavily used in the diagnosis of certain fungal
infections (coccidiomycosis or histoplasmosis).
(b) Disadvantage: These methods are labor-intensive and technically challenging.
(c) Advantage: Tests are very important in diagnosis of fungal infections.
iii) Western blot uses commercial membranes containing electrophoretically-separated
antigens, which are used to capture patient antibodies from serum. (Note that this is
‘backwards’ from western blotting you might have done in a research lab, where you
probably used known antibody to detect the presence of antigens). A primary use is in the
confirmatory testing of HIV infection.

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

Laboratory Diagnosis
ACTIVITY

1) Define pathognomonic, and look up an infectious disease which can be accurately diagnosed in
the absence of laboratory testing.

2) Which of the following test results would most likely be inaccurate if antibiotic treatment were
initiated prior to sampling?

a. G- diplococci visible in CNS specimen

b. Bacteriuria 1+
c. Blood cultures negative
d. Anti-spirochete antibodies not present
e. Gonococcal nucleic acid test negative

3) Order the following steps in laboratory diagnosis (some might occur at approximately the same
time, some might occur at different stages depending on the specific test):

a. Send interim report

b. Gram stain isolated colonies
c. Perform antibiotic susceptibility tests
d. Send preliminary report
e. Gram stain specimen
f. Perform biochemical tests (oxidase, catalase)
g. Read fermentation test results
h. Sandwich ELISA

4) How long would it take (approximately, in days) to get results from each of the following tests?
Would the result be given on the preliminary, interim, or final report?

a. Hepatitis serology panel

b. Antibiotic susceptibility
c. Oxidase
d. Fermentation
e. Gram stain of specimen
f. Acid-fast stain of specimen
g. PCR
h. Bacterial identification

5) What are 4 considerations, based on test metrics, when determining the importance to place on
a test result?

1.
2.
3.
4.

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

6) What type of microscopy would be required to visualize each of the following?

a. Auramine acid-fast stain

b. Spirochetes
c. Quellung
d. Silver stain
e. Indirect fluorescent test
f. Virus
g. Gram stain
h. Internal bacterial structure
i. Calcofluor white fungal stain
j. Bacillus sporulation
k. Ova

7) A medium for bacterial culture contains specific concentrations of NaCl, tryptone (tryptic digest
of casein), and sucrose. Is it a defined or complex medium, and why?

8) For each of the following scenarios, state whether a selective or differential culture approach
would be most useful:

a. Isolating a pathogen from an oral swab

b. Determining whether an isolate can ferment sorbitol
c. Isolating a suspected slow-growing pathogen
d. Determining susceptibility to bacitracin
e. Identifying Campylobacter

9) For each of the following components (not entire media), state whether it is selective,
differential, or both, how it works, and what it selects for and/or differentiates:

a. Neutral red in MacConkey agar

b. Crystal violet in MacConkey agar
c. Cetrimide in cetrimide agar
d. Potassium tellurite in tellurite agar
e. Bile salts in bile esculin agar
f. Methylene blue in eosin methylene blue agar
g. Salt in mannitol salt agar
h. Blood in blood agar
i. Lysed blood in chocolate agar
j. Antibiotics in Thayer-Martin agar
k. Ferric ammonium citrate in Hektoen enteric agar
l. Iron in bile esculin agar

10) What are 3 major differences between the processes to identify fungi and those to identify
bacteria?
a. Sample preparation:
b. Microscopic characteristics:
c. Media for culture:

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

11) Of the following methods, serological testing is most likely to be used to determine whether an
infection with an obligate intracellular pathogen has occurred. Why might each of the methods
below be inferior to serology in this case? Consider technical ease, availability, and methods for
detecting the analyte.

a. Culture in vitro
b. Observation of cytopathic effects
c. Fluorescent antibody staining
d. Growth in an animal model

12) Name a test that:

a. Looks for a pH change due to ammonia production

b. Looks for a pH change due to acid production
c. Cannot be positive for obligately-fermentative organisms
d. Will be positive for Staphylococcus aureus and some Yersinia sp., negative for
everything else
e. Depends on bacterial cell envelope structure
f. Requires overnight incubation
g. Is positive upon the production of oxygen
h. Depends on visualization of a precipitate
i. Looks for specific antigen in a patient’s specimen
j. Looks for specific antibodies in a patient’s specimen
k. Must be carried out on a specimen coming from a pure bacterial culture
l. Is positive for Escherichia but negative for Salmonella
m. Is positive for Salmonella but negative for Shigella

13) Serotyping is often done by mixing a suspension of isolated bacteria with multivalent antibodies
specific for surface structures (such as capsule). What type of test is this an example of? Draw
out how the structure that indicates a positive test is formed on a microscopic level.

14) Explain the difference between a direct (sandwich) ELISA and an indirect ELISA, both in terms
of process and intended analyte.

15) What is the primary difference between an indirect ELISA and an indirect fluorescent antibody
(IFA) test detecting the same thing?

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

16) What is the difference between the analytes identified by PCR and RT-PCR? Of these
techniques, which would you preferentially use to determine:

a. Presence of a bacterium

b. Presence of a herpesvirus

c. Presence of HIV

d. Expression of an exotoxin gene

e. Presence of an exotoxin gene

17) A patient is infected for the first time with an obligate intracellular organism which can produce
a proteinaceous cell wall. They have never been infected with any other obligately intracellular
organism. Draw a graph plotting antibody levels (dependent variable) against time
(independent variable). Time 0 indicates time of infection; antibody level 0 indicates levels
present prior to this infection. Draw curves for anti-rickettsial IgM, anti-rickettsial IgG, anti-
chlamydial IgM, and anti-chlamydial IgG. Indicate points on the curve that, when compared,
could provide 1) evidence of active infection, and 2) evidence of infection resolution.

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Fundamentals 2 Lab Diagnosis and Bacterial ID Dr. Drummelsmith

18) For each scenario, choose the types of diagnostic tests that would be needed to identify the
causative agent. If multiple test types are needed to obtain a result, determine what order you
would use them in. Consider speed, sensitivity, specificity, technical ability/infrastructure
needed, value of result, etc. Some cases might have several options, so think about this too,
and think about which method would be ideal and why. This is meant to get you to think
generally about these test types, not go look up specific tests. There may not be an absolutely
correct answer, although some are probably better than others!

Scenario Microscopy Culture Antigen Nucleic Acid Serology

An intestinal parasite with a
characteristic ovum
An intestinal parasite that appears
very similar to a common, non-
pathogenic commensal but has a
unique surface protein
A bacterium that will grow quickly
in vitro and has known nutritional
requirements
A bacterium that grows very slowly
in vitro and has known nutritional
requirements
A bacterium with unknown
nutritional requirements (i.e.
cannot be grown in vitro)
A bacterium that causes a rapidly-
progressing, severe disease
A very slow-growing bacterium for
which you need antibiotic
susceptibility data
A virus with highly characteristic
cytopathological effects
A virus producing common
cytopathological effects

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