Microbio Lab 4 Flashcards
smear
A thin film of material containing microorganisms spread over the surface of a clean microscope slide;Good for discerning (1) the morphology of cells such as rods, cocci, and other bacterial shapes; (2) the arrangement of cells, such as single cells, chains, or clusters; and (3) internal structures, such as endospores and cell inclusions
negative staining
Stains that are acidic and thus have a negatively charged chromophore that does not penetrate the cell but rater is repelled by the similarly charged bacterial cell; stains background; useful in studying the morphology of bacterial cells and characterizing some of the external structures, such as capsules, that are associated with bacterial cells; heat fixation is avoided to avoid shrinking the structures
capsule
extracellular gel-like layer (distinct and gelatinous) that occurs outside of the cell
protective structures because they prevent phagocytic white blood cells from engulfing and destroying the pathogen
allows attachment of organisms to solid surfaces in environment
slime layer
extracellular gel-like layer (diffuse and irregular) that occurs outside of the cell
glycocalyx
external layer made up of polysaccharides (‘sugar shell’)
capsular staining (Anthony method)
smears are prepared from cultures in grown in skim milk broth and air dried (bot not heat fixed); Stained with crystal violet for 2 minutes which binds cell and milk culture (background of plate) which causes capsule to appear white
Gram stain
Differential stain that differentiates from gram-positive cells and gram-negative cells
primary stain
initially stains both gram-positive and gram-negative cells (crystal violet)
mordant
forms an insoluble complex in gram-positive cells with primary stain (iodine)
decolorization
Removes the dye-mordant complex from gram-negative cells, leaving them colorless (ethyl alcohol)
peptidoglycan
Makes up the bacteria cell wall
composed of unique complex carbohydrate and protein
counterstain
Recolorizes the gram-negative cells (safarin)
gram-negative
Possesses a thin layer of peptidoglycan between a cell membrane and outer membrane
gram-positive
Possesses a thick layer of peptidoglycan and a cell membrane; no outer membrane
endospores
structure to survive harsh environmental conditions (exhaust essential nutrients); a dormant cell stage;
resistant to heat, drying, and numerous chemicals
hard outer covering of bacteria to protect themselves while dormant
when nutrients become available it goes through germination to form a new vegetative cell
autoclave
steam under pressure. kills all organisms and endospores. steam must contact items surface
spore staining (Shaeffer-Fulton)
Heat is applied while staining with malachite green, which penetrates the cell and becomes trapped in the endospore; safranin stains the vegetative portion of the cell
acid-fast staining (Ziehl-Neelsen)
Phenol and heat facilitate the penetration of the carbolfuchsin into the cell (heat acts as a mordant to make the mycolic acid and cell wall lipids more permeable to the stain); subsequent treatment of the cells with acid-alcohol, a decolorizer, does not remove the entrapped stain from cells; methylene blue is used as counter stain to visualize non acid-fast cells
acid-fast cells stained pink
Kinyoun acid-fast method
modification to acid-fast staining in which the concentration of primary stain, basic fuchsin and phenol are increased, making it unnecessary to heath the cells during the staining procedure
working stock culture
source of inoculum for various tests and stains
Goals to preparing a smear
- adhere the cells to the microscope slide so that they are not washed off during subsequent staining and washing procedures
- it is important to ensure that shrinkage of cells does not occur during staining, or distortion of artifacts can occur
- to prepare thin smears because the thickness of the smear will determine if you can visualize individual cells, their arrangement, or details regarding microstructures associate with cells.
How does smear preparation of cells from liquid medium differ from preparation of cells from a solid medium?
Cells from a liquid medium are placed using a few loopfuls of the broth and cells from a solid medium are smeared in a few drops of water on the slide.
Why is it important to limit the quantity of cells used to prepare a smear?
Too many cells on a slide can obscure the shape and arrangement of the cells as well as inhibit the identification of internal structures.
Describe the potential consequences of making a smear that is too thick.
Stain can become entrapped in the clumps of cells, preventing its removal by destaining and washing and leading to erroneous results for staining reactions
For preparation of a smear on a slide, what is the purpose of heat fixation? What problems can arise when the slide is heated in the flame?
Heat fixation kills the cells and fixes them to the slide so they don’t fall off. Overheating a slide could cause it to shatter.
Why is the gram stain considered a differential stain?
It differentiates between two types of bacteria based on the composition of the cell walls.
How do gram-positive and gram-negative bacteria differ in cellular structure, and how does this contribute to their differential staining properties?
Gram positive bacteria have thicker walls of peptidoglycan that traps crystal violet, making it purple. Gram negative bacteria have two thin cell walls with a thin layer of peptidoglycan between from which the stain is washed almost completely, making it pink after the counterstain.
How does the age of the culture affect the Gram stain reaction? What is an optimum age for a valid Gram reaction?
Old cultures of gram-positive cells may not retain stain as well as younger cultures and could give false negatives. Cultures that are 16-18 hours are best.
Which step in the Gram stain procedure is most prone to error? If done incorrectly, how might that step affect the end result?
The decolorization step
too much- gram positive cells lose their primary stain and become pink
too little- gram negative cells will not lose their primary stain and remain purple
What is the function of a mordant, and which reagent serves this purpose in the Gram stain procedure?
A mordant combines with the crystal violet and forms an inseparable complex in gram positive cells. Iodine in the reagent used.
List the reagents of the Gram stain technique in order and their general role in the staining process?
Primary stain (crystal violet) - stains cell walls
Mordant (iodine) - fixes stain in gram-positive cells
Decolorizer (ethyl alcohol) - removes stain from gram-negative cells
Counterstain (safranin) - colors gram negative cells pink
In what type of cell, gram-negative or gram-positive, would you find lipopolysaccharide in its cell wall?
gram-negative
reserve stock culture
backup culture; viable for several weeks; do not use to prepare stained slides or to make inoculations of various media
mycolic acid
complex lipid that is composed of fatty acids and fatty alcohols that have hydrocarbon chains and up to 80 carbons in length; prevents penetration of many stains
