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

• Collection of specimensProper method of collectionProper labeling of specimens

• Perform the diagnostic test• Feedback information to the Physician

Diagnostic Medical Microbiology

The physician should:

1.Inform the Laboratory of the clinical information and preliminary diagnosis

2.what laboratory examinations to request3.Know when and how to take the specimens4.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.

• 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.

Basic Issues in Proper Handling of Specimens

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?