Staining/gram staining quiz

gram positive

thick peptidoglycan cell wall

gram negative

thin peptidoglycan cell wall

have outer membrane composed of phospholipids and lipolysaccharide

general process of gram staining:

• crystall violet

• iodine

• alcohol

• counterstain

iodine/mordant:

forms complex w/ crystall violet, forms trapping agent

alcohol/acetone/decolorizer:

loses outer membrane of gram negative and crsytall violet iodine complexes are washed out, solubilizes lipids and acts as a dehydrating agent.

The success of Gram staining depends on the extent of the decolarization step

counterstain:

• safranin

• taken up by both cells

• colors gram negative red or pink

• gram positives, dark purple color is stronger so they just appear purple

what type of stain is gram staining?

differential

staphylococcus epidermidis

• gram positive control

• shape: coccus

• natural arrangement: staphylococcal clusters

• species: gram-positive

escherichia coli

• gram negative control

• shape: bacillus (rod-shaped)

• natural arrangement: single rods

• species: gram-negative

simple stains:

use of a single stain

• cation stains: positvely charged dyes that are attracted to negative cell wall and inside of cell including crystall violet of safranin

• negative stain: dye does not stain cells, surrounds cells instead, slide is not washed because cells repel stain causing them to appear as clear areas within the stain

differential stains:

• 2 or more dyes in combination

• take advantage of differences in cell structure

• ex: gram stain, acid-fast stain

structural stains:

• stain particular structures and not the whole cell

• identify presence or absence of structures in cells

• ex: endospore stain (malachite green), capsule stains, flagellar stains

what to do to underside of slide?

always clean it with a Kimwipe to remove debris

troubleshooting viewing stains:

• make sure slide is right-side up otherwise 100x lens will hit slide and u won’t be able to focus on cells

• only brightfield setting should be used

• make sure light isn’t too bright, first use knob on side then lever on sub-stage condenser

for gram staining using two cationic dyes:

• crystal violet

• safranin

• attracted to negatively charged cell surface and negatively charged biomolecules in cell

what does NOT stain with gram staining?

gram indeterminate organisms

what produces mixed results on gram staining?

gram variable organisms

decoloration issues:

• if gram negative is not left in decolorizer long enough, it will be purple

• gram positive that have been destained for too long will appear pink

Treponema Pallidum

• spirochaete can be stained using simple staining, identified by spiral shape

Mycobacterium leprae and M. tuberculosis

stained using acid-fast staining

Mycobacterium issues:

• have large amounts of lipid mycolic acid in their cell wall

• makes them waxy and hydrophobic

• resistant to decolorization

peptidoglycan cell walls?

porous and allow passage of small molecules through them, however it does slow the entry and exit of dyes

periplasmic space

the space between the cytoplasmic membrane and the outer membrane (or Gram positive cell wall)

what setting should Gram stained cells be observed with?

brightfield

effect of decolorizer on Gram positive vs. negative:

• Positive: the thicker cell wall of Gram positive bacteria keeps the CV-I complex from leaving the cell, alcohol shrinks the pores of the thick peptidoglycan layer

• Negative: cell membrane is dissolved, easily penetrates the peptidoglycan layer, interacts with the lipid of the cytoplasmic membrane, as it exits the cell it takes the CV-I complex with it

why is it important to dip the stains in water after the decolorization step?

it dilutes the decolorizer and stops the process of decolorization from continuing. if decolorization is halted it can remove stain from the Gram positive stain as well, it just takes longer

what do thick smears do to staining process?

make decolorization difficult, leading to uneven destaining

why should phase contrast never be used for Gram staining?

it will alter the perception of the colors on the stain

what type of cells does Gram staining work most reliably with?

young, actively growing cells. as cultures get older, the integrity of the cell walls become compromised

Mycobacterium have what sort of cell walls?

waxy and hydrophobic. Impermeable. Due to large amounts of lipid mycolic acid

why are mycobacterium called acid-fast?

because they are resistant to decolorization by acids

M. leprae:

Hansen’s Disease/leprosy

M. tuberculosis:

tuberculosis