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methods in diagnostic microbiology ppt.pptx
- 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: 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
- 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.
- 9. 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.
- 10. 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
- 11. 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
- 12. 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.
- 14. 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
- 17. Acid Fast • Physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures. • Ziehl-Neelsen Stain
- 18. 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.
- 20. 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
- 22. 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
- 23. Endospores
- 24. 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
- 25. 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
- 27. 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
- 28. Culture of Bacillus anthracis
- 29. Negative Stain • Uses Nigrosin
- 30. 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
- 31. 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.
- 32. • 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
Editor's Notes
- The physician on the other hand 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
- Micro organisms are though ubiquitous in their occurrence, common sources for their isolation are soils, lakes and river muds. Common techniques used for the isolation of industrially useful micro organisms include the following: (i) direct sponge of the soil; (ii) soil dilution; (iii) gradient plate method (pour plate and streak plate techniques); (iv)aerosol dilution; (v) flotation; (vi) centrifugation. Although for the detailed description of the above techniques, the readers are advised to consult a book on practical microbiology, they should recognize that the technique of isolating micro-organisms also varies according to the nature and physiological properties of the microbe to be isolated
- This method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique in 1882 and published it in 1884 to discriminate between two types of bacteria with similar clinical symptoms: Streptococcus pneumoniae (also known as the pneumococcus) and Klebsiella pneumoniae bacteria While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram variable and Gram indeterminant groups as well. Some organisms are Gram-variable (that means, they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and information-rich than differential staining.
- (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore color the cell.[9] When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell will lose its outer lipopolysaccharide membrane and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain will be removed from both gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink or red color.[10][11] Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative.[12] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain
- Acid-fast organisms are difficult to characterize using standard microbiological techniques (e.g. Gram stain - if you gram stained an AFB the result would be an abnormal gram positive organism, which would indicate further testing), though they can be stained using concentrated dyes, particularly when the staining process is combined with heat. Once stained, these organisms resist the dilute acid and/or ethanol-based de-colorization procedures common in many staining protocols—hence the name acid-fast
- The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria, is responsible for the staining pattern of poor absorption followed by high retention. The most common staining technique used to identify acid-fast bacteria is the Ziehl-Neelsen stain, in which the acid fast bacilli are stained bright red and stand out clearly against a blue background.
- An endospore is a dormant, tough, and non-reproductive structure produced by certain bacteria from the Firmicute phylum. The name "endospore" is suggestive of the bacterium changing internally to a spore or seedlike form (endo means within), but it's not a true spore (not an offspring). The endospore becomes important when the bacterium is experiencing an environment that is deleterious to the usual vegetative state of the bacterium, notably including when the bacterium is getting dried out (desiccated). Endospores enable the survival of a bacterium through periods of environmental stress. When the environment returns to favorable, the endospore can reactivate itself to the vegetative state. Not all, nor even most, types of bacteria can change to the endospore form. Examples that can include Bacillus and Clostridium. [1] The endospore consists of the bacterium's DNA and part of its cytoplasm, surrounded by a very tough outer coating. Endospores can survive without nutrients. They are resistant to ultraviolet radiation, desiccation, high temperature, and chemical disinfectants. Common anti-bacterial agents that work by destroying vegetative cell walls don't work on endospores. Endospores are commonly found in soil and water, where they may survive for long periods of time. Up to 15% of the dry weight of the endospore consists of calcium dipicolinate within the core, which is thought to stabilize the DNA. Dipicolinic acid could be responsible for the heat resistance of the spore, and calcium may aid in resistance to heat and oxidizing agents. Examples of bacteria having terminal endospores include Clostridium tetani, the pathogen that causes the disease tetanus. Bacteria having a centrally placed endospore include Bacillus cereus, and those having a subterminal endospore include Bacillus subtilis. Sometimes the endospore can be so large the cell can be distended around the endospore, this is typical of Clostridium tetani. Visualising endospores under the light microscope can be difficult due to the impermeability of the endospore wall to dyes and stains. While the rest of a bacterial cell may stain, the endospore is left colourless. To combat this, a special stain technique called a Moeller stain is used. That allows the endospore to show up as red, while the rest of the cell stains blue. Another staining technique for endospores is the Schaeffer-Fulton stain, which stains endospores green and bacterial bodies red. The arrangement of spore layers is as follows: Exosporium Spore coat Spore cortex Core wall
- Using an aseptic technique, bacteria are placed on a slide and heat fixed. The slide is then suspended over a water bath with some sort of porus paper over it, so that the slide is steamed. Malachite green is applied to the slide, which can penetrate the tough walls of the endosppores, staining them green. After five minutes, the slide is removed from the steam, and the paper towel is removed. After cooling, the slide is rinsed with water for thirty seconds. The slide is then stained with diluted safranin for two minutes, which stains most other microorganic bodies red or pink. The slide is then rinsed again, and blotted dry with bibulous paper.
- Optional - add 1 drop of lactophenol cotton blue
- For the purpose of gelling the microbial culture, the medium of agarose gel (Agar) is used. Agar is a gelatinous substance that is derived from seaweed. Microbiological cultures utilize petri dishes of differing sizes that have a thin layer of agar based growth medium in them. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated in an oven usually set at 37 degrees Celsius. Another method of bacterial culture is liquid culture, in which case desired bacteria are suspended in liquid broth, a nutrient medium. These are ideal for preparation of an antimicrobial assay. The experimenter would inoculate liquid broth with bacteria and let it grow overnight in a shaker for uniform growth, then take aliquots of the sample to test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides)
- Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores, appear light against the dark surrounding background. An alternative method has been developed using an ordinary waterproof marking pen to deliver the negative stain. Negative staining at both light microscope and electron microscope level should never be performed with infectious organisms unless stringent safety precautions are followed. Negative staining is a very mild preparation method and does not reduce the possibility of operator infection.
- 'Specimens submitted for microbiological testing require proper handling from the time of collection through all stages of transport, storage and processing. Issues common to all clinical specimens submitted for microbiological testing include not only proper identification, but also collection techniques that maximize recovery of microbiological pathogens and minimize contamination.
- Specimens must be collected with the use of strict aseptic techniques from anatomical sites most likely to yield pathogenic organisms. Specimens should be collected in such a way that contamination by indigenous flora is minimised. This is of vital importance for cultures of blood, bone or other tissues or fluids in which infection can be caused by indigenous flora and for specimens collected from sites of putative infection, that are contiguous to, or immediately adjacent to, cutaneous or mucosal surfaces. Sufficient material must be submitted.
- All general specimens should be transported in sterile specimen containers directly to the laboratory. If there is to be any delay in getting the specimen to the laboratory, appropriate transport media should be used. Specimens for culture should be transported to the laboratory as promptly as possible for processing. Unavoidable delays must be minimised. Most specimens can be transported at room temperature. Most specimens requiring prolonged storage before processing should be refrigerated. This maintains the viability of pathogens and preserves them in their relative proportions. This latter factor is crucial when semi-quantitative cultures are necessary for interpretation of results. Refrigeration also minimises the growth of contaminants.