Topic 10: Gram Staining

CV I

________ complex can not be washed out of gram- positive cells.

Application of primary stain (crystal violet)

all bacteria are stained purple by this basic dye

Application of mordant (grams iodine)

iodine combines with the crystal violet in the cell to form a crystal violet-iodine complex (CV-I)

Application of decolorizing agent (ehtyl alcohol or ethyl alcohol-acetone)

primary is washed out of some bacteria while others are unaffected

Application of secondary stain or counterstain (safranin)

basic dye stains the decolorized bacteria red

The gram stain is what type of stain?

differential stain

The gram stain allows the classification of bacteria as either ___ or ___.

gram positive or gram negative

Who discovered the gram staining technique?

Hans Christian Gram

What are the four basic steps of gram staining?

1. apply primary stain
2. apply mordant
3. apply decolorizing agent
4. apply secondary stain/counterstain

What is the primary stain used in gram staining?

crystal violet (basic dye)

After first step of gram stain, all the bacteria appear ___.

purple

What is the mordant used in gram staining?

gram's iodine

How is the CV-I complex formed?

iodine combines with the crystal violet in cell

What is the decolorizing agent used in gram staining?

ethyl alcohol or ethyl alcohol-acetone

After the second step of gram stain, what does the slide look like?

all bacteria still remain purple

After the third step of gram stain, what does the slide look like?

some bacteria remain purple while others are clear

What is the secondary stain/counterstain used in gram staining?

safranin (basic dye)

After the fourth step of gram stain, what does the slide look like?

bacteria remains purple or bacteria that was decolorized appear red/pink

What is the most important determining factor in the gram staining procedure?

that bacteria differ in rate of decolarization

Bacteria that decolorizes easily are ___.

gram negative

Bacteria that decolorize slowly are ___.

gram positive

Why does gram positive cells keep the purple stain throughout the gram staining procedure?

CV-I complex cannot be washed out

What occurs to gram negative cells that causes the loss of CV-I complex in cell wall?

decolorzing agent dissolve the outer lipopolysaccharide layer and CV-I complex washes out through thin layer of peptidoglycan

Gram stain is most consistent when done with ___ cultures of bacteria. (how old?)

young; less than 24 hrs

Why can't older bacteria be used for gram staining?

cell walls begin to degrade therefore may not retain primary stain and give inaccurate results

What is the first step in identifying bacteria?

gram staining