Microbiological Lab Techniques Flashcards
Purpose of microscopy
To visualize smallmicroorganisms (diagnostic microbiology)
Brightfield microscopy
Use of focused light to visualize small objects (objective x ocular lens = mag); example; wet mount
Darkfield microscopy
Scattered or reflected light to visualize object; visualization of thin bacteria (spirochete)
Fluorescent microscopy
One wavelength of light is used to cause another wavelength of light to be detected; fluorescent molecules target specific cell components
Electron microscopy
Electrons used to visualize; high mag; transmission = electrons through; scanning = electrons bounce
Purpose of direct examination
Visualize cells with minimal preparation
Wet mount
Direct visualization in water/saline; no processing (i.e. vaginal wet mount)
KOH
Usually 10% to dissolve proteinaceous non-fungal elements
Lactophenol cotton blue
Type of KOH added to non-specifically stain material (ex. fungal elements)
India ink
Type of KOH added to stain everything but yeast polysaccharide capsule (ex. cryptococcus)
Iodine
Stain organelles darker than cytoplasm (ex. parasite cysts)
Differential stain
Colored dyes that differentiate organisms or structures within organisms
Acid fast stain
Stains mycobacterium, nocardia, cryptosporidium
Fluorescent stains
Calcofluor white stain - interacts with cellulose and chitin in cell walls and fluoresces blue
Wright-Giemsa stain
differential used to stain parasites, intracellular bacteria, viral elements, and eukaryotic cells (polychromatic stain that contains azure B, methylene blue, and eosin Y; eosin ions stain basic structures orange/pink, others stain acidic structures blue/purple
Methenamine silver
Used to stain fungal elements and some bacteria in tissues
Trichrome stain
Used to detect fecal protozoa
Gram stain
1.) Heat fix
2.) Crystal violet solution - purple stain is positively charged; enters bacterial cell and complexes with anionic molecules
3.) Gram’s iodine - iodine acts as a mordant; interacts with crystal violet and forms a larger complex
4.) Gram’s decolorizer - alcohol dehydrates; peptidoglycan wall shrinks and captures crystal-violet iodine complex within cell
Gram + = thick cell wall doesn’t let crystal violet through
Gram - = thin cell wall allows stain through
5.) Safranin - red counter stain that stains whatever primary stain didn’t (Gram - )
Ziehl-Neelsen and Kinyoun stains
Same principle as gram stain but for mycobacterium; carbofushin (purple dye) added; alcohol washes what didn’t stain; methylene blue (green) acts as counter stain
Auramine-rhodamine
Fluorescent stain of mycolic acids
Purpose of culture medium
- select for growth of specific microorganisms
- differentiate amongst microorganisms
Nonselective culture
Supports growth of many organisms
Trypticase soy/broth agar
Digested soybean meal and casein; full of nutrients (supports growth of many organisms)
Nutrient broth/agar
Peptone and beef extracts; full of nutrients
Blood agar
Trypticase soy with sheep blood (enriched)
Chocolate agar
Trypticase soy with lysed sheep blood (enriched)
Thioglycolate broth
Sodium thioglycolate added to promote a reducing (anaerobic) environment; used to grow anaerobic bacteria and can be used to differentiate oxygen usage
Sabouraud dextrose agar
Growth of fungi
Selective culture; differential
Supports growth of specific organisms usually by limiting growth of others; differentiation of organisms in a mixture
MacConkey agar
Selective for gram- ; differential for lactose fermentation
Mannitol salt agar
Selective for gram+ Staphylococci; differential for S aureus (mannitol fermentation)
Lowenstein-Jensen agar
selective for mycobacteria species
Eosin-methylene blue agar
Selective for gram- ; differential for lactose fermentation
Hektoen enteric agar
Selective for gram - ;differential for hydrogen sulfide production (black) and lactose/sucrose/salicin formation (pink)
Specialized culture
Ingredients support the growth of specific organisms
Thayer martin
Neisseria gonorrhoeae
Buffered charcoal yeast extract (BCYE) agar
Legionella
Thiosulfate citrate bile salts sucrose (TCBS) agar
Vibrio species
Regan-Lowe agar and Bordet-Gengou agar
Bordetella pertussis
Purpose of biochemistry in lab techniques
Differentiation of bacteria based on varying biochemical pathways
Catalase test
Bubbles = + catalase; catalase enzyme functions as part of the antioxidant defense response; breaks down H2O2 to O2 and 2H2O
Oxidase test
Purple = positive (before 30 secs); some bac use O2 as final electron acceptor (reduce O2 to form H2O via cytochrome oxidase complex)
Antibiotic sensitivity test (Kirby-Bauer, Disk Diffusion)
Wells with increasing amounts of antibiotics are inoculated with clinical sample; resistant = will grow, sensitive = no growth; first well in an antibiotic series with no growth is the minimum inhibitory concentration
Purpose of immunology in labs
Use of immune components (usually antibodies) to differentiate microorganisms
Examples of immunology in lab
Latex agglutination, immunofluorescence, ELISA, western blot, and others
Latex agglutination
- Latex bead solution (with antibody coating) added to flat surface
- mix bacterial strain in
- evaluate for precipitation (precipitate = +)
Immunofluorescence
Use of antibodies that are conjugated to fluorescent molecules
Purpose of eukaryotic cell culture
Grow intracellular bacteria and viruses; eukaryotic cells act as the culture medium for intracellular bac and viruses
