differential staining

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which of the three differential stains would likely be the first used when identifying an unknown bacterium? why?

almost all bacteria can be differentiated by gram stain into the two groups whereas only a very small percentage of bacterial species are either spore formers or acid fast

what is the function of a mordant?

it causes the primary stain to adhere better or be taken up by the cell so that it is not removed during the decolorizing step

for differential staining, how does a counterstain differ from a primary stain?

a counterstain must be a different color than the primary stain to aid differentiation

how do gram positive and gram negative bacteria differ in cellular structure? how does this contribute to their differential staining properties?

a gram positive cell, which has a thick cell wall, retains the crystal violet dye better in the presence of a decolorizer as compared to a gram negative cell, which has a thin wall

which is the most critical step in the gram stain procedure? why? if this procedure is done incorrectly, how might this affect the results?

the decolorizer step is the most critical because it is the step in which the cells become differentiated. if too much is used, the gram positive cells will lose the primary stain and be counterstained pink. if too little is used, gram negative cells will not lose the primary stain and will remain purple

how does culture age affect the results of a gram stain?

old cultures of gram positive cells may not retain stain as well as younger cultures and could give false negative results

how does culture age affect the results of a spore stain?

old cultures of spore formers are ideal because under the conditions of nutrient depletion sporulation is more likely to occur

why must smear thickness be considered before performing a gram stain?

smears that are too thick will not stain properly because the stains may not penetrate lower layers and thick clumps of cells may not be easily decolorized

what color are bacterial endospores after a gram stain? what does this tell you about the physical properties of endospores?

basic dyes do not penetrate spores so gram staining will result in colorless spores. this indicates that spores are very resistant structures

bacillus anthracis, the causative agent of anthrax, is an endospore former. why does this trait enhance its capabilities as a biological weapon?

bacillus anthracis spores are ideal for biological weaponry because they are easily produced, can be dispersed in the air and are environmentally stable

what makes mycobacterium particularly resistant to staining? how are the bacteria in the genus grouped in terms of gram classification?

it has a peptidoglycan layer filled with mycolic acids that make the cell wall waxy and impenetrable to stains. they are classified with gram positive cells because of cell wall thickness and genetic similarities

how do you think the acid fast nature of mycobacterium contributes to its virulence?

the waxy cell wall of mycobacterium protects the bacterium against phagocytosis and some antibiotics while in the host so the pathogen has greater opportunity to cause disease

gram stain

crystal violet primary stain, iodine mordant, alcohol or acetone decolorizer, safranin counterstain, purple positive, pink negative

spore stain

malachite green primary stain, heat and water mordant, rinsed water decolorizer, safranin counterstain, green positive, pink negative

acid fast stain

carbolfuchsin primary stain, heat mordant, acid-alcohol mordant, methylene blue counterstain, red positive, blue negative

bacterial cell wall is composed of:

peptidoglycan

the exosporium, or endospore coat, is composed of:

proteins

endospores are produced by bacteria in the genus:

bacillus and clostridium

acid fast staining is useful for identifying the causative agent of:

leprosy and tuberculosis

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