Microscopy, Staining, and Direct Examination of Specimens PDF

MICROSCOPY, STAINING, AND DIRECT EXAMINATION OF SPECIMENS Phase Contrast Microscopy MICROSCOPY Utilizes beams of light passing through the specimen...

MICROSCOPY, STAINING, AND DIRECT EXAMINATION OF SPECIMENS Phase Contrast Microscopy MICROSCOPY Utilizes beams of light passing through the specimen Most common method used both for the detection of that are partially deflected by the different densities microorganisms directly in clinical specimens or thickness of the microbial cells or cell structures in Used for the characterization of microorganism grown the specimen in culture Does not use fixed smear To determine presence of an infection Direct observation of unstained specimens in a wet To characterize organisms grown in culture preparation or wet mount To visualize etiologic agents (bacteria, yeast, parasites) not readily seen by the naked eye alone Fluorescent Microscopy To screen specimen adequacy (iBartlett's classification) Utilizes fluorophores or fluorochromes that can be raised to a higher energy level after absorbing UV light Principal Factors that Influence the Quality of These dye molecules return to their normal, lower Microscopic Image energy state where they release excess energy in the 1. Magnification form of visible (fluorescent) light Producing an image of the desired size, larger than Fluorochroming Direct chemical interaction with the the original object component of the bacterial cell ➔ Total magnification – obtained by multiplying the (Acridine orange, Auramine, Calcofluor eyepiece magnification by the objective magnification white) ➔ Low power = 10 x 10 = 100 x total magnification ○ 400 x (HPO) ; 1000x (oil immersion) Immunofluorescence Antibodies are conjugated to a ○ We need immersion oil so that the light won’t fluorescent dye (Fluorescein isothiocyanate / FITC) scatter the image or so it won’t refract the light 2. Resolution Fluorescing object appears bright against a dark Extent to which detail in the magnified object is background maintained Excitation/Barrier filter – prevents the excitation Resolving power – the shortest distance between two wavelengths from damaging the observer’s eyes; selects points that can be determined to be separate objects and limits the wavelength of transmitted light Dependent on numerical aperture of the objective lens Digital Photography – often used to maintain 3. Contrast permanent records The ability to find and see the specimen surface or Ziehl-Neelsen stained Auramine-Rhodamine stained internal structural details distinctly Simplest method for altering contrast – stain the specimen to enhance and distinguish the details Can be adjusted through the diaphragm Types of Microscopy Bright-Field (Light) Microscopy Principle: visible light is passed through the specimen - specimen’s image appears dark against a Dark-Field Microscopy brighter background Involves alteration of microscopic technique rather Magnification of up to 1000x (objective lens x ocular than the use of dyes or stains to achieve contrast lens) allows for the visualization of fungi, most The condenser does not allow light to pass directly parasites and bacteria through the specimen but directs the light to hit the To achieve the level of resolution desired with 1000x specimen at an oblique angle magnification, oil immersion must be used in ○ Specimen appears luminous against a dark conjunction with light microscopy background ○ Oil immersion – used to fill the space between the Used for detecting bacteria with thin dimension such objective lens and glass slide as spirochetes (ex. Treponema pallidum) Upright, inverted and stereomicroscopes – all utilize Limited to specialized research facility bright-field microscopy Modern microscopes are parfocal and parcentric Electron Microscopy Uses electrons instead of light to visualize small objects where these electrons are focused by electromagnetic fields and form an image on a fluorescent screen Allows magnification up to 10,000,000x 2 general types: 1. Transmission electron microscope (TEM) 2. Scanning electron microscope (SEM) 1  Digital Automated Microscopy AKA Digital or virtual microscopy It uses sophisticated software and unique technology to acquire microscopic digital images of Gram stains using a web interface Uses a fully automated microscope to be viewed on a single screen Can greatly aid in the delivery of a quality cost-effective microscopy training DIRECT AND INDIRECT SMEARS Direct Smear A preparation of the primary clinical sample received in the laboratory for processing Directly from patient sample/specimen Indirect Smear Primary sample has been processed in culture and the 2. Differential Stains smear contains organisms obtained after purification Divides bacteria into separate groups or growth on artificial media Directed towards coloring components of those Preparations from solid, semisolid media or broth elements present 1. A growth from a solid medium is transferred to the Principal stain utilized in bacteriologic examination surface of slide using an inoculating loop or needle Provides rapid presumptive identification of pathogens 2. Emulsified in a drop of sterile water/saline on slide and assessment of specimen quality 3. Allow to air-dry and affixed to the slide by placing it Bacteria will retain the stain based on the characteristics on a slide of its cell wall Thick cell walls (peptidoglycan) – retain the crystal violet-iodine (CV-I) complex Ex: Gram staining & Acid-Fast Bacilli (AFB) staining Steps in differential staining: 1. Application of primary stain The sample from a liquid medium (broth culture) is 2. Application of mordant applied to the slide, air-dried and fixed before staining 3. Application of decolorizing agent 4. Application of secondary stain/counterstain 3. Diagnostic Antibody/DNA probe-mediated Staining Directed specifically at identification of an organism Used for the specific identification of selected pathogens, such as Chlamydia trachomatis, Bordetella The smear is examined for the presence of pertussis, Legionella pneumophila, HSV, varicella-zoster microorganisms, white and red blood cells, epithelial virus, CMV, adenoviruses and respiratory viruses cells, and mucus 4. Negative Staining Examination of smears can provide rapid information by Used as wet mounts visualization of patient specimens. Used to demonstrate the presence of diffuse capsule ○ It can also be used to detect different surrounding some bacteria microorganisms present in the same specimen The bacteria appear as light-colored bodies against a May provide a clue to the type of infection and if dark background – the cell surface repels the acidic additional workups are indicated stain since the bacterial cells are negatively charged Used for evaluating patient specimens for the presence Example: India ink or Nigrosin dye (acidic stain) of cells indicative of inflammation or contamination STAINING TECHNIQUES Staining methods are used: 1. To visualize the contents of the smear 2. To differentiate one group of organisms from another group of organisms 3. To identify organisms via staining the special structure of the organism Basic dyes – commonly used dyes in Microbiology ○ These are cationic or have positively charged groups that bind to negatively charged molecules like nucleic acids and proteins ○ Examples: methylene blue, crystal violet, safranin GRAM STAINING Types of Stains Principal stain used for microscopic examination of 1. Simple Stains bacteria Single stain is used Provides a mechanism for the rapid presumptive Directed towards coloring forms and shapes present identification of pathogens Examples: malachite green, methylene blue, crystal Based on the chemical differentiation of organism as a violet, carbolfuchsin, safranin result of the structural chemical components of the organism’s cell wall 2  7. Toxin-producing organisms such as clostridia, staphylococci, and streptococci – may destroy white blood cells within a purulent specimen 8. Faintly staining gram-negative organisms, such as Campylobacter and Brucella – may be visualized using an alternative counterstain (e.g., basic fuchsin) Exception in Gram Staining Those organisms that exist almost exclusively within host cells (chlamydia) Gram-Positive Those that lack cell wall (mycoplasma & ureaplasma) Cell walls contain thick peptidoglycan with numerous Those of insufficient dimension to be resolved by teichoic acid cross-linkages that contribute to ability of light microscopy (spirochetes) organisms to resist decolorization ACID-FAST STAIN ○ Cell wall retains the primary dye following Used to stain bacteria whose cell walls contain application mordant, gram’s iodine (CV-I complex) long-chain fatty (mycolic) acids Gram-Negative Mycobacteria – most commonly encountered acid-fast Cell walls consist of a thinner layer of peptidoglycan bacteria and the presence of an outer lipid bilayer Has some degree of acid-fastness – Nocardia spp., (lipopolysaccharide) that is dehydrated during coccidian parasites (Cryptosporidium spp.) decolorization Principle: the primary stain binds to mycolic acid in the ○ The CV-I complexes are not trapped within the cell walls of the mycobacteria and is retained after peptidoglycan decolorization with acid alcohol ○ Acid-fast bacilli (AFB) retain primary stain and are colored red & non-AFB blue or green (methylene blue or malachite green counterstains) color 2 methods: 1. Ziehl-Neelsen Method Classic acid-fast method Requires heat to allow the primary stain (carbolfuchsin) to enter the waxy, mycolic acid-containing cell wall 2. Kinyoun Acid-Fast Method (Cold Method) Has higher concentration of phenol in the primary stain solution All cocci are Gram-positive ; except Neisseria, AFB Non-AFB Branhamella and Veillonella, Megasphaera All bacilli are Gram-negative ; except Arachnia, Primary stain (Carbol fuchsin) Red Red Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Erysipelothrix, Eubacterium Kurthia, Lactobacillus, Listeria, Propionibacterium, Mordant (ZN method: heat) Red Red Mycobacterium, Actinomycetes, Nocardia, Rhodococcus, Gordonia and Tsukamurella Decolorizer (Acid-alcohol) Red Colorless Counterstain (Methylene blue) Red Blue Limitations of Gram Staining 1. Over-decolorization – may result in the identification of false gram negative results, whereas under-decolorization may result in the identification of false gram-positive results 2. Smears that are too thick or viscous – may retain too much primary stain, making the identification of proper Gram stain reactions difficult ○ Gram-negative organisms may not decolorize properly 3. Cultures older than 16 to 18 hours – will contain living and dead cells Modified Acid-Fast Staining Method Useful for the identification of oocysts of the ○ Cells that are dead will be deteriorating and will not coccidian species (Cryptosporidium, Cytoisospora and retain the stain properly Cyclospora) 4. Stain may precipitate with aging Specimen: concentrated sediment of fresh or ○ Filtering through gauze will remove excess crystals formalin-preserved stool 5. Gram stains from patients on antibiotics or Reagents: same with the conventional acid-fast antimicrobial therapy – may have altered gram stain reagents except for the concentration of the acid reactivity because of the successful treatment alcohol (1% H2SO4) 6. Pneumococci identified in the lower respiratory tract Results: oocysts appear as magenta-stained organism on a direct smear – will not grow in culture against a blue/green background ○ Some strains are obligate anaerobes 3  2. Endospore Stain Dorner (Carbol fuchsin & Nigrosin dye) Schaeffer-fulton (Malachite green & Safranin) ○ Observation of intracellular refractile bodies ○ Heating and alcohol treatment allows penetration and decolorization of stain FLUORESCENT STAINS (Fluorochromes) 1. Auramine-Rhodamine Used to stain mycobacteria Binds to the mycolic acid of the organism’s cell wall Result: yellow to orange bacteria with dark green background 3. Flagella Stain Gray (Carbolfuchsin & Tannic Acid) Leifson (Carbol fuchsin, Tannic Acid & Methylene Blue) ○ Unstable colloidal suspension of tannic acid salts are used to form precipitate on the cell walls and flagella – increased diameter and allows staining 2. Acridine Orange Selectively binds to any nucleic acid Useful in demonstrating small amounts of bacteria in blood culture, CSF, urethral smears and other exudates Useful in staining bacteria w/o cell wall (mycoplasma) 3. Calcofluor White Binds to the cell walls of fungi Binds to cells that are composed of chitin or cellulose 4. Nucleic Acid Stain Feulgen (Carbol fuchsin) ○ Specific for DNA Acridine orange FUNGAL STAINS ○ Binds all nucleic acid 1. Lactophenol cotton blue ○ Fluorochrome ○ Stains the chitin in the fungal cell walls ○ Useful in staining mycoplasma ○ Made up of lactic acid (preservative), phenol (inhibitory to other fungus), cotton blue (stain- chitin) 2. Methenamine silver stain ○ Used in histology to stain fungal elements in tissue specimens 3. Periodic acid-Schiff (PAS) ○ Used to stain molecules with a high carbohydrate content ○ Preferred stain for direct clinical material ANTIBODY-CONJUGATED STAINS Immunofluorescence uses an antibody conjugated to a fluorescent dye Used for the direct examination of patient specimens where the dye will bind to the corresponding antigen if it is present in the specimen Used to directly examine patient specimens for bacteria that are difficult or slow to grow (e.g. Legionella spp. and Chlamydia trachomatis) Can also be used in virology SPECIAL STAINS 1. Capsule Stain Anthony’s, Hiss’s and Gin’s (Crystal Violet) Nigrosin (Nigrosin dye) Welch (Crystal Violet) ○ Treatment with hot crystal violet solution and followed by a copper sulfate solution rinse 4