Microbiology Staining
Gram stain - answer Positive differential stain, primary dye: crystal violet, mordant:
iodine, decolorizer: acetone alcohol, counter stain: safranin, gram positive: purple, gram
negative: pink.
Simple stain - answer Positive stain methylene blue, blue cells, shape/arrangement
apparent.
Acid fast stain - answer Positive differential stain, primary dye: Carbol fuchsin, mordant:
steam, decolorizer: acid alcohol, counter stain: methylene blue, acid fast: red/pink, non-
acid fast: blue.
Endospore stain - answer Positive differential stain, primary dye: malachite green,
mordant: steam, decolorizer: dH2O, counter stain: safranin, endospores: green,
vegetative cells: pink.
Negative stain - answer Negative stain (acidic/anionic dyes), nigrosine, gray cells on a
dark background (shape and size apparent).
Simple stain - answer Single staining reagent is used to color the bacterial smear.
Simple stain - answer Common dyes are crystal violet and methylene blue.
Electrical charge - answer All bacterial cells are slightly negatively charged because of
the large numbers of phospholipids that compose the cell membranes. This negative
charge is attracted to the positively charged chromosomes or basic dyes.
Simple stain procedure - answer1. Disinfect bench top, gather materials.
2. Prepare smear.
3. After smear has air dried, heat fix.
4. Flood smear with methylene blue and allow to stand 1 minute.
5. Wash gently with distilled water.
6. Place slide within bibulous paper or paper towel and gently blot dry.
7. Observe with brightfield microscope.
Why do we use basic dyes to stain bacterial cells? - answer Bacterial cells have a
negative charge which binds to the positively charged stain.
Why don't acidic dyes stain bacterial cells? - answer Acidic stains have a colorless
cation, such as sodium, and a colored anion. The positively charged sodium will not
, cause a noticeable color change when bonded to the cell wall. When rinsed, the colored
part will wash away.
Most important differential stain used in microbiology - answer Gram stain
Differential stain - answer Make use of two dyes, the primary dye applied first, and the
counter stain applied at the end of the procedure.
Gram-reaction method - answer Used to differentiate bacterial species into gram-
positive and gram-negative, based on the chemical and physical properties of their cell
walls.
Gram-positive cells - answer Have a cell wall composed primarily of a thick layer of
peptidoglycan.
Gram-negative cells - answer Composed of two layers. The outermost layer called the
outer membrane composed of a phospholipid’s bilayer. The outer membrane covers a
thin layer of peptidoglycan generally 1/4 to 1/10 the thickness seen in the gram- positive
cell wall.
Crystal violet - answer Primary stain, stains both gram-positive and gram-negative cells
purple.
Decolorization - answer Involves application of alcohol which causes the dehydration of
the peptidoglycan of gram-positive cells to make it more permeable to the crystal violet-
iodine complexes. The effect on gram-negative cells is quite different; the alcohol
dissolves the outer membrane of these cells and even punches small holes in the thin
layer of peptidoglycan layer that allows the crystal violet-iodine complexes to exit the
cells. The cells have been differentiated into gram-positive, which appear purple; and
gram-negative, which are colorless. To see the gram-negative we apply the counter
stain, safranin, where the gram-negative cells would appear pink/red.
Gram stain procedure - answer1. Prepare smear
2. Flood surface of smear with crystal violet and allow to stand for 1 minute. (No need to
rinse)
3. Tilt slide to allow excess crystal violet to run off and flood smear with iodine. Allow to
stand 1 minute.
4. Tilt slide at 45-degree angle and allow excess crystal violet-iodine mixture to run off.
Keeping slide tilted, add drops of decolorizer onto surface of slide for no longer than 15
seconds.
5. Wash slide with dH2O to remove all decolorizer.
6. Flood surface of slide with safranin and let stand for 1 minute.
7. Rinse safranin and blot dry.
8. Examine under microscope using oil immersion.
Gram stain - answer Positive differential stain, primary dye: crystal violet, mordant:
iodine, decolorizer: acetone alcohol, counter stain: safranin, gram positive: purple, gram
negative: pink.
Simple stain - answer Positive stain methylene blue, blue cells, shape/arrangement
apparent.
Acid fast stain - answer Positive differential stain, primary dye: Carbol fuchsin, mordant:
steam, decolorizer: acid alcohol, counter stain: methylene blue, acid fast: red/pink, non-
acid fast: blue.
Endospore stain - answer Positive differential stain, primary dye: malachite green,
mordant: steam, decolorizer: dH2O, counter stain: safranin, endospores: green,
vegetative cells: pink.
Negative stain - answer Negative stain (acidic/anionic dyes), nigrosine, gray cells on a
dark background (shape and size apparent).
Simple stain - answer Single staining reagent is used to color the bacterial smear.
Simple stain - answer Common dyes are crystal violet and methylene blue.
Electrical charge - answer All bacterial cells are slightly negatively charged because of
the large numbers of phospholipids that compose the cell membranes. This negative
charge is attracted to the positively charged chromosomes or basic dyes.
Simple stain procedure - answer1. Disinfect bench top, gather materials.
2. Prepare smear.
3. After smear has air dried, heat fix.
4. Flood smear with methylene blue and allow to stand 1 minute.
5. Wash gently with distilled water.
6. Place slide within bibulous paper or paper towel and gently blot dry.
7. Observe with brightfield microscope.
Why do we use basic dyes to stain bacterial cells? - answer Bacterial cells have a
negative charge which binds to the positively charged stain.
Why don't acidic dyes stain bacterial cells? - answer Acidic stains have a colorless
cation, such as sodium, and a colored anion. The positively charged sodium will not
, cause a noticeable color change when bonded to the cell wall. When rinsed, the colored
part will wash away.
Most important differential stain used in microbiology - answer Gram stain
Differential stain - answer Make use of two dyes, the primary dye applied first, and the
counter stain applied at the end of the procedure.
Gram-reaction method - answer Used to differentiate bacterial species into gram-
positive and gram-negative, based on the chemical and physical properties of their cell
walls.
Gram-positive cells - answer Have a cell wall composed primarily of a thick layer of
peptidoglycan.
Gram-negative cells - answer Composed of two layers. The outermost layer called the
outer membrane composed of a phospholipid’s bilayer. The outer membrane covers a
thin layer of peptidoglycan generally 1/4 to 1/10 the thickness seen in the gram- positive
cell wall.
Crystal violet - answer Primary stain, stains both gram-positive and gram-negative cells
purple.
Decolorization - answer Involves application of alcohol which causes the dehydration of
the peptidoglycan of gram-positive cells to make it more permeable to the crystal violet-
iodine complexes. The effect on gram-negative cells is quite different; the alcohol
dissolves the outer membrane of these cells and even punches small holes in the thin
layer of peptidoglycan layer that allows the crystal violet-iodine complexes to exit the
cells. The cells have been differentiated into gram-positive, which appear purple; and
gram-negative, which are colorless. To see the gram-negative we apply the counter
stain, safranin, where the gram-negative cells would appear pink/red.
Gram stain procedure - answer1. Prepare smear
2. Flood surface of smear with crystal violet and allow to stand for 1 minute. (No need to
rinse)
3. Tilt slide to allow excess crystal violet to run off and flood smear with iodine. Allow to
stand 1 minute.
4. Tilt slide at 45-degree angle and allow excess crystal violet-iodine mixture to run off.
Keeping slide tilted, add drops of decolorizer onto surface of slide for no longer than 15
seconds.
5. Wash slide with dH2O to remove all decolorizer.
6. Flood surface of slide with safranin and let stand for 1 minute.
7. Rinse safranin and blot dry.
8. Examine under microscope using oil immersion.
