Lab - Stains

Tools for staining

inoculating loop, bunsen burner, empty slides, sterile water, staining kit, bacteria culture plates

Bacteria cell walls

most have a negative charge on their cell wall

Basic stains

consists of positively charged chromogen / chromophore (dye) that is attracted to or absorbed into the negatively charged bacteria cell walls

• also called direct stains or cationic stains

Acidic stains

repelled by the bacteria cell wall and stain the background and leave the cell transparent (called negative stains)

• also called indirect stains or anionic stains

Simple stain

• uses only a single dye

• reveals shape, size, and cell arrangements

Making a smear

1. place a few paper towels down, mark top of slide with T, put clothespin over T

2. place drop of sterile water on slide, take a small amount of bacteria, and let air dry

3. swipe slide over flame to heat fix

Basic stain steps

1. cover top of slide with stain for 30-60 seconds (crystal violet or safranin)

2. rinse stain off with water

3. blot with paper towels

4. view using oil immersion lens

Negative stain steps

1. put down paper towels

2. put a drop of nigrosin stain at one end of the slide

3. transfer loopful of culture to stain and mix well

4. take a second slide and place it on at 30 degree angle and pull back until it touches the drop

5. push second slide back across the slide

6. wait 5 mins to air dry

Differential stains

• uses different colored dyes (a primary dye and a counter stain)

• can distinguish cell types (gram(+) or gram(-)

• can highlight parts of the cells (capsules)

• examples: gram, acid-fast, and endospore stains

Gram negative

stain pink

Gram positive

stain purple

Red cells

acid-fast

Blue cells

not acid-fast

Gram stain

• developed by Hans Christian Gram in 1884

• consists of crystal violet (primary), iodine (mordant), an alcohol rinse (decolorizer), and safranin (counterstain)

• different results are due to differences in the structure of the cell wall and how it reacts to the series of reagents applied to the cells

• remains the universal basis for bacterial classification and identification

• a practical aid in diagnosing infection and guiding drug treatment

Gram positive cell walls

traps the stain in the envelope above the membrane

• contains teichoic acid and lipoteichoic acid

Gram negative cell walls

stain is rinsed from first membrane with decolorizer

• 2 phospholipid bilayers

Gram stain steps

1. sterilize loop, apply sterile water drop, mix in loopful of culture, heat fix

2. apply crystal violet (stains everything purple), wait 10 seconds, rinse with water

3. apply Gram’s iodine (mordant), wait, rinse with water

4. apply alcohol (decolorizer), wait, rinse with water

5. apply safranin (red dye), wait, rinse, view

Crystal violet

stains both gram (+) and gram (-) purple

Gram’s iodine

a stabilizer that causes the dye to form large complexes in the peptidoglycan meshwork of the cell wall; thicker cell walls are able to more firmly trap the large complexes

• mordant

• gram (+) = dye trapped in wall

• gram (-) = no effect

Alcohol

application of alcohol dissolves lipids in the outer membrane and removes the dye from the peptidoglycan (only in gram negative cells)

• gram (+) = crystals remain in cell wall

  • gram (-) = outer membrane weakened, wall loses dye

Safranin

gram negative bacteria are colorless after decolorization, their presence is demonstrated by applying the counterstain’

• gram (+) = red dye masked by violet

• gram (-) = red dye stains the colorless cell

Things that cause false gram stains

• gram variability

• smear thickness

• under decolorization

• over decolorization

• heat fixing

Gram variability

as gram-positive cultures age, cells die and their cell walls break down and can no longer retain the crystal-violet iodine complex

• if most of the organisms on your slide are gram-positive you still consider these as positive

Smear thickness

if your smear is too thick it can give you a false result

Under decolorization

not leaving the decolorizer on long enough can prevent gram-negative cells from taking up the counterstain

• false gram-positive

Over decolorization

leaving the decolorizer on too long can wash out all stains

• false gram-negative

Heat fixing

if you heat fix for too long, it can damage cell walls and prevent them from retaining the crystal violet-iodine complex

• false gram-negative

• start over if smear is brown