Methods in Diagnostic

Microbiology
Nanette Ramilo-Cruz, MD, DPAFP
 Case
• 82/M was brought to the ER due to 1 week
history of weakness, fever, productive cough
and anorexia.
• HX: Admitted at hospital 1 week PTC due to
COPD and HPN
• PE: Temp 39c RR 26 HR 110 BP 130/80
: crackles RLF, base
• Diagnosis?
•Laboratory exams? Specimens needed?
•Laboratory Procedures used to identify the
microbial agent?
 Objectives
• Discuss the functions of a Diagnostic Medical
Microbiology
• Discuss the common laboratory methods
used in the diagnosis of infectious diseases
• Enumerate the common biological specimens
used in the diagnosis of infections diseases
• Discuss the proper method of collection,
handling, storage of these biological
specimens
 Diagnostic Medical Microbiology
• Concerned with the etiologic diagnosis of
infection
• encompasses the characterization of
thousands of agents that cause or are
associated with infectious diseases.

• Clinical Information Diagnosis

Lab Test
 Diagnostic Medical Microbiology
• Collection of specimens
Proper method of collection
Proper labeling of specimens
• Perform the diagnostic test
• Feedback information to the Physician
The physician should:

1.Inform the Laboratory of the clinical

information and preliminary diagnosis
2.what laboratory examinations to request
3.Know when and how to take the specimens
4.How to interpret the results
 Common Biological Specimens
• Blood/serum
• Sputum/bronchial washings
• Exudates/transudates
• Urine and other body fluids
• Feces
• Swabs of tissue samples
 Microbial Isolation
• Direct sponge
• Dilution
• Gradient plate method (pour plate and streak
plate techniques)
• Flotation
• Centrifugation.
 Laboratory Methods
(1) Direct Microscopy: Morphologic identification of
the agent in stains of specimens or sections of
tissues
(2) Culture isolation and identification of the agent.
(3) Biochemical Tests:
A. Detection of antigen from the agent by
immunologic assay (latex agglutination, EIA, etc)
or by fluorescein-labeled (or peroxidase-labeled)
antibody stains.
 Laboratory methods
B. DNA-DNA or DNA-RNA hybridization to detect
pathogen-specific genes in patients'
specimens.
C. Demonstration of meaningful antibody or
cell-mediated immune responses to an
infectious agent
 Gram Staining

• 1882 – Hans Christian Gram

• Differentiate bacterial species into two large
groups (Gram-positive and Gram-negative)
based on the chemical and physical properties
of their cell walls
 Gram Stain
• Gram-positive bacteria: thick mesh-like cell
wall made of peptidoglycan (50-90% of cell
wall), which stains purple
• Gram-negative bacteria: have a thinner layer
(10% of cell wall), which stains pink. Outer
membrane contains lipids, and is separated
from the cell wall by the periplasmic space.
 Basic Steps in Gram Stain
• Heat-fix bacterial smear
• Apply the Crystal Violet
• Apply Gram’s Iodine
• Rapid decolorization with Alcohol/ acetone
• Counterstain with Safranin
Gram positive Bacteria
Gram Negative Bacteria
 Acid Fast
• Physical property of some bacteria referring
to their resistance to decolorization by acids
during staining procedures.
• Ziehl-Neelsen Stain
 Ziehl-Neelsen Stain
• Cover with tissue paper
• Flood slide with carbolfuchsin, the primary
stain, for 2 minutes while heating with steam
or heating on hot plate.
• Remove paper cover, decolorize slide with a
mixture of hydrochloric acid and ethanol.
• Counter stain with methylene blue.
 Notable Acid Fast Structures
• All Mycobacteria - M. tuberculosis, M. leprae,
M. smegmatis and atypical Mycobacterium
• Nocardia
• Head of sperm
• Bacterial spores
• Parasites like Cryptosporidium parvum
Isospora and Cyclospora cysts
Acid Fast Bacteria
 Schaeffer-Fulton Stain
• Isolate endospores
• Stains endospores green, and any other
bacterial bodies red.
• The green stain is malachite green,
• Counterstain is safranin, which dyes any other
bacterial bodies red
Endospores
 Potassium Hydroxide Test (KOH)
• Detects fungi
• Dissolve human cells. KOH denatures the
proteins in the human cell; only the fungal
cells remain to be seen under the microscope.
• Athlete's foot, fungal vaginitis and many other
fungal infections
 KOH Test Procedure
• Take scraping from margin (not center) of
lesion
• Place on clean slide
• Add 2-3 drops of 10% KOH in water
• Warm the slide (don't boil)
• Add cover slip
• Examine immediately under high dry
magnification with light microscope
 Microbial Culture
• Method of multiplying microbial organisms by
letting them reproduce in predetermined
culture media under controlled laboratory
conditions
• Importance: Diagnostic Purposes
Prognosis of disease
• Using: Agar
Culture of Bacillus anthracis
 Negative Stain
• Uses Nigrosin
 Proper Handling of Microbial
Specimens
• Very Important!
• crucial for obtaining microbiological test
results that are both timely and clinically
relevant.
• Maximizes Cost-effectiveness of laboratory
test
 Basic Issues in Proper Handling of
Specimens
• Collection of Specimens
• Important information includes:
* the specific site(s)
* whether the patient was receiving antibiotics prior to
collection
* specific pathogens that are being sought
* the methods by which the specimen was collected
* whether patient may be infected with pathogens
known to be dangerous to laboratory staff.
 Basic Issues in Proper Handling of
Specimens
• Transport of Specimens
• Storage of specimens
• Specimens that should not be refrigerated include:
* blood--should be left at room temperature or in an
incubator at 5[degrees]C
* cerebrospinal fluid--transport at room temperature
* Neisseria species--transport rapidly to the
laboratory.
QUESTIONS?
 Case
• 82/M was brought to the ER due to 1 week
history of weakness, fever, productive cough
and anorexia.
• HX: Admitted at hospital 1 week PTC due to
COPD and HPN
• PE: Temp 39c RR 26 HR 110 BP 130/80
: crackles RLF, base
• Diagnosis?
•Laboratory exams? Specimens needed?
•Laboratory Procedures used to identify the
microbial agent?