Chapter 8 Smears and Stains (MICRO)

kylerigonan's version from 2016-03-03 04:44


Question Answer
what are the advantages of wet mount slides?can see living microbes, observe motility, see true cell shape, dimensions
what are the disadvantages of wet mount slides?microbes frequently are colorless, lack contrast with liquid in which they are suspended, therefore hard to see
How do you "fix" a microscope slide?smear a thin film of microbes on surface of a clean slide. Flame fixed. Then slide can be immersed in alcohol to fix the smear
What is flame fixing?Sliding pass a few times through a flame to attach the cells to the slide
What are the advantages of fixing cells to slide?Prevents cells from washing away when stained, Preserves cells (fixed smears may be stored in lockers for days before staining). Kill microbes. May increase dye uptake by breaking down cell membranes
What are the disadvantages of fixing cells to slide?Distortion of cell shape and size. Inability to determine motility as organism is dead.
How do stains work?Most stains and dyes are salts that ionize when in aqueous solution. One ion will carry a pigment molecule, the "colored" portion of the dye.
What is the chromophore?color carrier ion
What are cationic chromophoresBasic dyes that carry a positively charged colored ion.
What are anionic chromophores?Acidic dyes that carry a negatively charged ion.
Why is pH important for the staining process?At 7 pH or higher, most cells carry a net negative charge from ionized carbohydrates and proteins. The cations (basic) will bind with negative charge
What are simple stains?Procedures that used single dyes
What are the pros and cons of simple stains?Simple stains allowed someone to determine the size, shape, and arrangement of cells, but could not differentiate between types of bacteria (based upon cell wall composition) nor identify specific structures such as endospores or flagella.
What is the pros of using a differential stain?use more than one dye and can distinguish between different types of bacteria or can differentiate different structures within a bacterium/cell. The most famous differential stain is called the Gram Stain developed by Danish pathologist, Han Christian Gram in 1884
Gram-positive bacteria appears ______purple
Gram-negative bacteria appearsRed/pink
What is a counterstain?After decolorization, a second contrasting red basic dye, safranin, is added. Only the gram-negative cells would have "available" negative charges for binding to the safranin and appears red/pink
Both gram-positive and gram-negative bacteria are initially stained by the complex of ____ & _____. How do they interact?crystal violet (primary stain) and Gram's iodine (acts a mordant by combining with crystal violet and forms large complexes that are trapped within the cell and the PG complex of cell walls)
why is alcohol/acetone used in differential stain?alcohol/acetone acts as a decolorizing solution and acts as a lipid solvent
what does alcohol/acetone do to gram-negative bacteria?It destroys the cell membrane and outer membrane of gram-negative bacteria. The thin layer of PG is too thin to trap the crystal violet-iodine complexes so it washes out as cell is decolorized. ("then stained by red safranin counterstain)
what does alcohol/acetone do to gram-positive bacteria?The thick layers of cell wall PG collages, trapping the crystal violet-iodine complexes within the cell resulting in purple color
Fungal cells stain gram-______positive; even though they lack PG.
why do Fungal cells stain gram-positive?Fungi have thick cell walls rich in chitin that is a thick, multi-layered polysaccharide-rich cell wall and is essential for gram-positive reaction.
Why should human/animal cells stain gram-negative when gram-stained?because they lack cell walls.
What is the importance of Gram stains in patient care?useful in the dx of bacterial pneumonia, bacterial bladder infections, gonorrhea, and pyogenic infections of the brain, lung, abdomen, pelvis and wounds. Information regarding antibiotic selection.
What might a gram-negative bacteria in a patient be useful?Gram-negative bacterial cell walls inhibit passage of some antibiotics such as penicillin, and penicillin is avoided for treatment of most gram-negative bacterial infections.
What is a false gram negative reaction?occurs when bacterial cells that have a gram-positive cell wall appear pink/red following the gram-stain procedure.
What may be a reason for a false gram negative reaction?old cultures that old PG begin to breakdown through bacterial enzymes called autolysins and over-decolorization.
what is an example of a false gram positive reaction? Why might it occur?If a bacterial cells with a gram-negative cell wall stain purple then false gram positive. D/t under-colorization and smears that are too thick
what are gram variable reactions?some of the cells appear red, others appear purple. *If pure, gram variable reactions suggest some of your cells are giving false reactions
Why is important to use young cultures of bacteria (less than 24 hours of age)?As gram-positive bacteria age, enzymes breakdown the thick layers of PG in the cell wall. When they breakdown the cell walls cannot trap crystal violet-iodine complexes and subsequently, old gram-positive bacteria will appear gram variable or gram negative ("false gram-negative" reaction)
What feature do some bacteria have that may hinder staining?Some stain-resistant bacteria had mycolic acid in their cell walls and were generally impermeable to many hydrophilic substances (including antibiotics and disinfectants)
Interaction between mycolic acids and chromophores? What do you NOT stain it with?Cell wall of mycolic acid in their cell walls are "waxy" HYDROPHOBIC lipids that do not attract chromophores of basic dyes. NOT stained with basic dyes
What are acid-fast bacteria (AFB's)?resist decolorization with acidified alcohol once they are stained with carbolfuchsin
What is the importance of knowing if bacteria is a AFB?Their cell wall protects against many antibiotics and disinfectants and prevent dessication (extreme dryness). They can survive for long time.