Sessions 2 & 3: Gram-Staining and Bacterial Culture

what is a smear preparation?

an emulsified bacterial sample

allows visualization of uniformly dispersed individual bacterial cells (microscopically)

what does a smear preparation consist of?

small drop of water

sample from bacterial colony

how is a smear prepared?

should be air-dried (prevents aerosol, prevents change in morphology due to boiling)

bacterial film that forms must be heat-fixed to surface of the slide (ensures bacteria won’t be washed away during staining, will stain better, sample is killed)

what must the slant culture be mixed with?

must be mixed with water

what must the agar plate be mixed with?

must be mixed with water

what must the broth culture be mixed with?

does NOT need to be mixed with water

why should you not use too much inoculum?

cells will aggregate

will have difficulty visualizing individual cells

what kind of stain is a Gram stain?

differential staining procedure

usually 1st differential test performed on a clinical specimen

divides bacteria into two groups: gram (+) and gram (-)

why do specimens reactly differently to gram staining procedure?

due to their chemical and physical differences in wall composition

what are the characteristics of gram (+) cell wall?

thick cell wall with sugars, amino acids, and peptidoglycan

peptidoglycan = polymer of cross-linked sugars (GlcNAc, MurNA)

allows cells to retain crystal violet-iodine complex

what are the characteristics of gram (-) cell wall?

thin cell wall with little peptidoglycan

have outer membrane (bilayered)

LPS (lipopolysaccharide) → composed of 3 parts: Lipid A, Core polysaccharide (Kdo), O-antigen

what are the main parts of the gram stain procedure?

primary stain

mordant

decolorizing agent

counterstain

what is the primary stain for gram stain?

crystal violet (basic dye)

(30 seconds)

what is the mordant for gram stain?

Gram’s iodine

(iodine complexes with crystal violet; 30 seconds)

what is the decolorizing agent for gram stain?

80% acetone/20% ethanol

(gram (+) will not decolorize,

gram (-) will decolorize and become colorless)

(~8-10 seconds)

what is the counter stain for gram stain?

Safranin O (basic dye)

(will stain any colorless, gram (-) cells and will not affect the color of gram (+) cells)

what shows when the gram stain is over-decolorized?

gram (+) bacteria will appear gram (-)

what shows when the gram stain is under-decolorized?

gram (-) will appear gram (+)

what happens to the gram stain when culture turns too old?

gram (+) can turn gram (-) if the culture is too old