Principles of Diagnostic medical microbiology

Diagnostic medical microbiology is concerned with the etiologic diagnosis of infection. Laboratory procedures used in the diagnosis of infectious disease in humans include the following: (I) Specimen (II) Microscopy & morphology (III) Cultivation (culture) (IV) Biochemichal reactions (V) Serological identification (VI) Molecular identification & typing (I) Specimen: The specimen must be obtained from the site most likely to yield the agent at that particular stage of illness Common types of specimens: Blood: in sepsis, bacteraemia, or toxaemia. Stool: in lower GIT infections. Urine: in urinary tract infections. CSF: in cases of meningitis. Pus: in any purulent infection. Swab: in infections involving accessible mucous membranes, such as: oral, nasal, pharyngeal, urethral, vaginal, or cervical swab. Aspirate: From fluids in body cavities, such as: joint, peritoneal fluid, or pleural effusion aspirate. Sputum: in lung infections (i.e. pneumonia). Punch biopsy: from skin infections. Biopsy: from other body tissues. (II) Microscopy & morphology When bacterial infection is suspected, most specimens are smeared on glass slides, Gram-stained, and examined microscopically. Common types of stains for suspected bacterial infections are:

- Gram stain: The most important differential stain used for

diagnostic identification of various organisms. - Ziehl-Neelsen stain (acid fast stain prepared by heating), or the less commonly used Kinyoun stain (acid fast stain prepared by cold technique); both are used to stain mycobacteria. - Immunofluorescent antibody (IF) stain in which the microorganism or its antigens are injected into an animal, in order to obtain antibodies from its serum, these antibodies are labeled by fluorescein and then added to the specimen, then the specimen is examined by special microscope for Ag-Ab reaction, which indicates presence of the specific organism. Common types of stains for suspected fungal or parasitic infections are: Calcofluor white Methenamine silver Periodic acid-Schiff (PAS)

(III) Cultivation (culture)

Simple media: Peptone water Nutrient broth Nutrient agar

Enriched media: For fastidious bacteria that requires presence of highly nutritive substances to grow. Blood agar Chocolate agar Loffler's serum

Selective media: These are media that contain substances that inhibit all except few types - of bacteria. Lowenstein Jensen (LJ) medium Blood tellurite Thayer Martin medium Desoxycholate citrate agar (DCA) TCBS Selenite broth Differential (indicator) media:

These are media that contain substances that change visibly due to metabolic activities of particular organisms. MacConkey's medium Cystine lactose electrolyte deficient (CLED) Triple sugar iron (TSI) agar Xylose lysine deoxycholate (XLD)

(IV) Biochemical reactions Sugar fermentation Indole production Voges-proskauer's reaction Methyl red test Urease test Catalase test Oxidase test DNase test Commercial kit systems Automated bacterial identification systems

(V) Serological identification The term serology refers to the use of Ag-Ab reaction. If a bacterium is not identified, then it is mixed with several prepared antisera, each containing a specific antibody, the antiserum in which agglutination occurs indicates the unidentified bacterium. Antiserum means serum that contains antibody or more antigens; it is prepared from the blood of animals inoculated parenterally with an antigenic material. The common methods of serological identification: Immunofluorescence (direct & indirect) Enzyme-linked immunosorbent assays (ELISA) Latex agglutination test Immunoblotting (western blot)

(VI) Molecular identification & typing: Molecular diagnosis simply means identification of the microbial nucleic acid. The common methods of molecular diagnosis are: Nucleic acid probes Polymerase chain reaction (PCR) Chromosomal DNA Restriction Endonuclease Analysis Plasmid profile analysis

How to answer diagnosis question in the exam? Cover the 6 items mentioned above, as follows: 1. Specimen: Mention the type of specimen. 2. Microscopic morphology: Type of stain. Gram positive or negative. Shape (cocci, bacilli, coccobacilli, ). Arrangement (chains, pairs, ). 3. Culture:

Type of agar. Temperature of incubation. Type of gas medium (O2, Co2, vaccum, ). Colonies morphology. Cultural characteristics (e.g. Bacitracin sensitivity).

4. Biochemical reactions: May be mentioned with culture. 5. Serological diagnosis 6. Molecular diagnosis