DIAGNOSTIC

MICROBIOLOGY
BIOCHEMICAL TESTS, IMMUNOASSAYS
AND MOLECULAR DIAGNOSTICS

Lecture 6
Aida L. Mamorno BSMT, M.S. Bio
Nikki Heherson A. Dagamac, M.Sc. and Reuel M. Bennett M.Sc.
ISOLATION OF PATHOGENS FROM
CLINICAL SPECIMENS
• Proper sampling and
culture of a suspected
pathogen is the most
reliable way to identify an
organism that causes a
disease

• Most clinical samples are

first grown on general-
purpose media, media such
as blood agar that support
the growth of most aerobic
and facultatively anaerobic
organisms.
• Enrichment culture, the use of selected culture media and incubation
conditions to isolate microorganisms from samples, is an important part
of clinical microbiology.
• Differential media are specialized media that allow identification of
organisms based on their growth and appearance on the media.
 GROWTH-DEPENDENT
IDENTIFICATION METHODS

• Traditional methods for identifying pathogens depend on observing

metabolic changes induced as a result of growth. These growth-
dependent methods provide rapid and accurate pathogen
identification.
 Tests To Know
 Case Study Tests
 Indole
 Methyl Red/Voges Proskauer
 Citrate
 H2S production in SIM
 Urea hydrolysis
 Motility
 Lactose fermentation
 Sucrose fermentation
 Glucose fermentation & gas production
 Staphylococcus identification tests
 BAP
 MSA
 Mstaph broth
 Coagulase
 Indole Test
 How to Perform Test: Inoculate Tryptone broth with inoculating
loop.
 Property it tests for: This test is performed to help differentiate
species of the family Enterobacteriaceae. It tests for the bacteria
species’ ability to produce indole. Bacteria use an enzyme,
tryptophanase to break down the amino acid, tryptophan, which
makes by-products, of which, indole is one.
 Media and Reagents Used: Tryptone broth contains
tryptophan. Kovac’s reagent—contains hydrochloric acid,
dimethylaminobenzaldehyde, and amyl alcohol—yellow in color.
 Reading Results: Kovac’s reagent reacts with indole and creates
a red color at the top part of the test tube.
Indole
 Methyl Red/Voges Proskauer
(MR/VP)
 How to Perform Tests: Inoculate 2 glucose broths
with inoculating loop. After 48 hours of incubation, add a
few drops of MR to one tube, and VP reagents to the
other tube.
 Properties they test for: Both tests are used to help
differentiate species of the family Enterobacteriaceae.
 MR—tests for acid end products from glucose fermentation.
 VP—tests for acetoin production from glucose fermentation.
 Media and Reagents Used:
 Glucose Broth
 Methyl Red indicator for acid
 Voges Proskauer reagents—A: 5% Alpha-Naphthol, & ethanol, B:
Potassium Hydroxide, & Deionized Water.
 MR/VP continued
 Reading Results:
 MR— a + result is red (indicating pH below 6) and a – result is yellow
(indicating no acid production)
 VP—A + result is red after VP reagents are added (indicating the
presence of acetoin) and a – result is no color change.

Methyl Red: left – and right + VP: left + and right –

 Citrate
 How to Perform Test: Inoculate slant with inoculating loop.
 Property it tests for: This test is used to help differentiate
species of the family Enterobacteriaceae. It is selective for bacteria
that has the ability to consume citrate as its sole source of carbon
and ammonium as sole nitrogen source.
 Media and Reagents Used: Simmon’s Citrate Agar contains
sodium citrate (carbon source), ammonium ion (nitrogen source), &
pH indicator—bromthymol blue.
 Reading Results:
 A + result is blue (meaning the bacteria metabolised citrate and
produced an acid end product) and a – result remains green
Citrate

Left positive and right negative.

 H2S Production in SIM
 How to Perform Test: Stab SIM
media with inoculating needle.
 Property it tests for: This test is used
to help differentiate species of the family
Enterobacteriaceae. This test is used to
determine the ability to reduce sulfur into
H2S.
 Media and Reagents Used: SIM
media contains the sulfur containing
amino acid, cysteine, sodium thiosulfate,
& peptonized iron or ferrous sulfate.
 Reading Results: H2S will react with
the iron or ferrous sulfate and produce a
black precipitate. A positive result has a
black precipitate present and a negative
result has no black precipitate.
 Urea Hydrolysis
 How to Perform Test: Inoculate Urea broth with
inoculating loop.
 Property it tests for: This test is done to
determine a bacteria’s ability to hydrolyze urea to
make ammonia using the enzyme urease.
 Media and Reagents Used: Urea broth contains
a yeast extract, monopotassium phosphate, disodium
phosphate, urea, and phenol red indicator.
 Reading Results: Urea broth is a yellow-orange
color. The enzyme urease will be used to hydrolyze
urea to make ammonia. If ammonia is made, the
broth turns a bright pink color, and is positive. If test is
negative, broth has no color change and no ammonia
is made.
 Motility Test
 How to Perform Test: Stab motility media with
inoculating needle.
 Property it tests for: This test is done to help
differentiate species of bacteria that are motile.
 Media and Reagents Used: Motility media contains
tryptose, sodium chloride, agar, and a color indicator.
 Reading Results: If bacteria is motile, there will be
growth going out away from the stab line, and test is
positive. If bacteria is not motile, there will only be
growth along the stab line. A colored indicator can be
used to make the results easier to see.
Motility

From left to right:

+ – +
 Lactose Fermentation
 How to Perform Test: Inoculate lactose broth with inoculating
loop.
 Property it tests for: This tests for the bacteria’s ability to
ferment lactose.
 Media and Reagents Used: Lactose broth contains beef
extract, gelatin peptone, and lactose. A phenol red indicator is
added to indicate acid production from fermentation.
 Results
 A positive result is yellow after indicator is added (indicating
lactose fermentation)
 A negative result will have no color change or will be redish.
 Sucrose Fermentation
 How to Perform Test: Inoculate sucrose broth with inoculating
loop.
 Property it tests for: This test is done to help differentiate
species of the family Enterobacteriaceae. This tests for the bacteria’s
ability to ferment sucrose and production of acid end-product
 Media and Reagents Used: Sucrose broth contains beef
extract, gelatin peptone, and sucrose. Phenol red indicator is added
to indicate an acid end-product.
 Results
 A positive result is yellow after indicator is added (indicating
sucrose fermentation)
 A negative result has no color change or is reddish.
 Glucose Fermentation & Gas
Production
 How to Perform Test: Inoculate broth with inoculating loop.
 Property it tests for: This test is done to help differnetiate
species of the family Enterobacteriaceae. This tests for the bacteria’s
ability to ferment glucose and produce gas and/or an acid end-
product..
 Media and Reagents Used: Glucose broth contains beef
extract, gelatine peptone, and glucose. A phenol red indicator is
added to indicate an acid enproduct. A Durham tube is added to
indicate gas production.
 Results
 A positive result for acid is yellow after indicator is added
(indicating glucose fermentation)
 A positive result for gas is a bubble in the Durham tube.
 A completely negative result has no color change or reddish color
and no bubble.
Sugar Fermentation Tests

Tube 1: Negative acid /Negative gas

Tube 2A: Must incubate longer (ambiguous result)
Tube 2B: Positive acid /Negative gas
Tube 3A: Positive acid/ Positive gas
 Mannitol Salt Agar (MSA)

 How to Perform Test: Inoculate an MSA plate using streak

plate method and incubate 24-48 hours.
 Property it tests for: This tests for the bacteria’s
ability to tolerate 7% salt concentration and ferment
mannitol. The media is selective because it selects for
salt tolerant bacteria. The media is also differential
because it differentiates the salt tolerant organisms on
their ability to ferment mannitol.
 Media and Reagents: MSA media contains nutrient
agar, mannitol, 7% sodium chloride and phenol red
indicator.
 MSA Results
 Reading Results:
 If the organism is tolerant to salt
it will grow.
 If the organism is not tolerant to
salt it will not grow.
 If the salt tolerant organism can
Growth with no mannitol fermentation.
ferment mannitol then there will
be yellow zones around the
colonies.
 If the salt tolerant organism
cannot ferment mannitol then
the media will remain pink.

Growth with + mannitol fermentation.

 Coagulase

 How to Perform Test: Inoculate rabbit plasma with

one single colony. Break up colony and stir until blended
in plasma. Incubate at 37 degrees C for 24 hours.
 Property it tests for: This tests for the
bacteria’s ability to clot blood plasma using the
enzyme coagulase. If the organism has
coagulase it will clump rabbit plasma.
 Media and Reagents: This media contains
rabbit plasma dissolved in buffer.
 Coagulase Results
 Reading Results:
 If the organism is has coagulase it will clump the plasma.
 If the organism does not have coagulase it will not clump the plasma.
ANTIMICROBIAL DRUG
SUSCEPTIBILITY TESTING
• Antimicrobial drugs are widely used for the treatment of infectious
diseases.

• Pathogens should be tested for susceptibility to individual antibiotics

to ensure appropriate chemotherapy. This rigorous approach to
antimicrobial drug treatment is usually applied only in health care
settings.

• The standard procedure that assesses antimicrobial activity is called

the Kirby–Bauer method (Figure 24.8).
• Agar media are inoculated by evenly spreading a defined density of a
suspension of the pure culture on the agar surface. Filter paper disks
containing a defined quantity of the antimicrobial agents are then
placed on the inoculated agar.

• Antibiograms are periodic reports that indicate the susceptibility of

clinically isolated organisms to the antibiotics in current local use.

• After a specified period of incubation, the diameter of the inhibition

zone around each disk is measured. Table 24.4 presents zone sizes for
several antibiotics.
SAFETY IN THE MICROBIOLOGY
LABORATORY
• Safety in the clinical laboratory requires effective training, planning,
and care to prevent the infection of laboratory workers with pathogens.

• Materials such as live cultures, inoculated culture media, used

hypodermic needles, and patient specimens require specific precautions
for safe handling.
IMMUNOASSAYS FOR INFECTIOUS
DISEASE
• Specific immune
responses,
particularly antibody
titers and skin tests,
can be monitored to
provide information
about past infections,
current infections,
and convalescence
POLYCLONAL AND MONOCLONAL
ANTIBODIES
• Polyclonal and monoclonal antibodies are used for research and
clinical applications.

• Hybridoma technology provides reproducible, monospecific antibodies

for a wide range of clinical, diagnostic, and research purposes.
AGGLUTINATION
• Direct agglutination tests are widely used for determination of blood
types (Figure 24.15).
FLUORESCENT ANTIBODY-BASED
METHODS

• can be used for identification, quantitative enumeration, and sorting

of a variety of cell types.
ENZYME-LINKED IMMUNOSORBENT
ASSAY AND RADIOIMMUNOASSAY
• ELISA and RIA methods are the most sensitive immunoassay
techniques.

• Both involve linking a detection system, either an enzyme or a

radioactive molecule, to an antibody or antigen, significantly enhancing
sensitivity.

• ELISA and RIA are used for clinical and research work; tests have been
designed to detect either antibody or antigen in many applications.
IMMUNOBLOT PROCEDURES
• Immunoblot (Western blot) procedures are used to detect antibodies
to specific antigens or to detect the presence of the antigens
themselves.
MOLECULAR AND VISUAL
DIAGNOSTIC METHODS
Nucleic Acid Hybridization
Polymerase Chain Reaction
(PCR)
• Perhaps the most widespread use of probe-based technology is in the
application of gene amplification (PCR) methods.

• Employs the used of a thermocycler.

•Various DNA-based methodologies are currently used in clinical, food,

and research laboratories.
DIAGNOSTIC VIROLOGY
• Virus propagation in vitro can be accomplished only in tissue culture.
Therefore, most diagnostic techniques for viral identification are not
growth-dependent but routinely rely on immunoassays and nucleic
acid–based techniques.

• Electron microscopy techniques are useful for direct observation of

viruses in host samples.