Microbio Lab Quiz (5.1)

Smear

1) Specimen is spread on a slide

2) Allow specimens to air dry

3) fixing

• heat fixing

• chemical fixing (alcohol)

fixing

prevents material from washing off the slide during the staining process

differential staining

uses two different-colored dyes

• cell components react differently tot he dyes

• used to distinguish cell types or parts

• ex: gram stain, acid-fast stain, and endospore stain

differential stain steps

1) Primary dye

2) decolorizer

3) Counterstain

primary dye

stains all the cells

decolorizer

removed dye form some types of cells

counterstain

stains the cells that had their dye removed

no effect on cells that retained the primary dye

gram stain

separates bacteria based on differences in their cell wall compostion

gram positive

thick layer of peptidoglycan

gram negative

thin layer of peptidoglycan with an outer membrane

gram positive bacteria

call walls that contain multiple layers of peptidoglycan (60%-90%)

molecules of teichoic acid within the layers of peptidoglycan

• teichoic acid may strengthen the cell wall

retain the primary dye

gram positive cell wall

gram negative bacteria

cell wall is thinner

contain a thin layer of peptidoglycan (10%-20%) and no teichoic acid

have an outer membrane of lipopolysaccharides

primary dye removed by decolorizer

pick up counterstain

gram negative cell wall

gram stain procedure

Step 1) Slide is covered with crystal violet for 1 minute

• all bacteria are purple

Step 2) Slide is covered with iodine for 1 minute

• Large complex forms with the crystal violet and the iodine

All bacteria are purple

Step 3) Slide is covered with Gram’s alcohol (quick rinse)

• gram positive cells remain purple

• alcohol dissolves the lipopolysaccharide layer in the Gram-negative cell wall (Crystal violet washes out)

gram negative cells are now colorless

Step 4) Slide is covered with safranin for 1 minute

• Gram positive cells are still purple

• gram negative cells now red

Gram Variable Reactions

bacteria that stain both purple and red

due to:

• age of culture

• technique

• genetics

• media

positive

cocci

staphylococcus

1) identify the grams stain reaction

2) identify this bacterial shape

3) arrangement

negative

bacilli

single, random, no specific arrangement

1) identify the grams stain reaction

2) identify this bacterial shape

3) arrangement