Micro Lab 3--Preparation of Bacterial Smears/Simple Staining/Negative Staining/Gram Stain

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Heat Fixation

performed by the rapid passage of air-dried smear two or three times over the flame of the bunsen burner

confluent

flowing or running together

simple staining

the bacterial smear is stained w/a single reagent, which produces a distinctive contrast between the organisms and its background. it is used to show the morphology and arrangement of bacterial cells, most commonly used:
methylene blue
crystal violet
carbol fuchsin

Cocci

are spherical in shape

Diplococcus

a pair of cocci

Streptococcus

a chain of cocci

Staphylococcus

a cluster of cocci

Tetrad

packet of 4 cocci

Sarcina

packet of 8 cocci or more

Bacilli

are rod shaped

Diplobacillus

a pair of bacilli

Streptobacillus

a chain of bacilli

Spiral bacteria

are rigid or flexible

Vibrios

are curved rods

Spirilla

are helical and rigid

Spirochetes

are helical and flexible

Negative Staining

requires the use of an acidic stain such as India ink or nigrosin, it stains around the bacteria because it cannot penetrate the cell wall, it colors the slides background leaving the organisms transparent. "negative copy" of a picture

Differential Staining

requires the use of at least three chemical reagents that are applied sequentially to a heat-fixed smear. the first reagent is called the primary stain. the second one is a decolorizing agent, final the counterstain

primary stain

its function is to impart its color to all cells

Decolorizing Agent

it may or may not remove the primary stain from the entire cell or only from certain cell structures (due to the cellular composition)

Counterstain

cannot be absorbed and the cell or its components will retain the color of the primary stain. if the primary stain is removed, the decolorized cellular components will accept and assume the contrasting color of the counterstain.

Gram Stain

it divides bacterial cells into two major groups, gram-positive and gram-negative, which makes it an essential tool for classification and differentiation of microorganisms.

Crystal Violet (Hucker's) (Primary Stain)

this stain is used first and stains all cells purple

Gram's Iodine (Mordant)

this reagent serves not only as a killing agent but also as a mordant, a substance that increases the cells' affinity for a stain. it does this by binding to the primary stain, thus forming an insoluble complex. the result: crystal-violet-iodine (CV-I) complex serves to intensify the color of the stain. cells appear purple-black

Ethyl Alcohol, 95% (Decolorizing agent)

serves a dual function as a protein-dehydrating agent and as a lipid solvent, its action is determined by two factors, the concentration of lipids and the thickness of the peptidoglycan layer in bacterial cell walls. in gram-negative cells it increases the porosity of the cell wall by dissolving the lipids in the outer layers. thus CV-I can be easily removed from the thinner and less highly cross-linked peptido layer, the washing-out effect facilitates the release of the unbound CV-I, leaving the cell colorless. the much thicker peptido layer in gram-positive cells is responsible for the more stringent retention of the CV-I complex, as the pores are smaller, due to the dehydrating affect of the alcohol. it is dificult to remove, cells remain purple.

Safranin

the final reagent, used to stain pink those cells that have been previously decolorized. since only gram-negative cells undergo decolorization, they may now absorb the counterstain. gram-positive cells retain the purple color of primary stain.

gram-veriable

organisms that are older than 24 hours tend to become this, they lose their ability to retain the primary stain and appear to be GV, some cells will appear purple while others appear pink

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