methods in diagnostic microbiology ppt.pptx

Methods in diagnostics microbiology

1. 1 South Kazakhstan Medical Academy DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY SUBMITTED BY IBRAR YASIN. SUBMIT TO . GROUP 14 A. TOPIC: Diagnostic methods in microbiology.

2. 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

3. 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

4. Collection of specimens Proper method of collection Proper labeling of specimens Perform the diagnostic test Feedback information to the Physician Diagnostic Medical Microbiology

5. The physician should: Inform the Laboratory of the clinical information and preliminary diagnosis what laboratory examinations to request Know when and how to take the specimens How to interpret the results

6. Common Biological Specimens Blood/serum Sputum/bronchial washings Exudates/transudates Urine and other body fluids Feces Swabs of tissue samples

7. Microbial Isolation Direct sponge Dilution Gradient plate method (pour plate and streak plate techniques) Flotation Centrifugation.

8. Laboratory Methods 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.

9. 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

10. 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

11. 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.

12. 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

13. Gram positive Bacteria

14. Gram Negative Bacteria

15. Acid Fast Physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures. Ziehl-Neelsen Stain

16. 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.

17. 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

18. Acid Fast Bacteria

19. 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

20. Endospores

21. 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

22. 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

23. 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

24. Culture of Bacillus anthracis

25. Negative Stain Uses Nigrosin

26. 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

27. 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.

28. 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