Lab Exercise 2 - Microscopy and Staining

What is staining useful for?

making bacteria easier to view by providing contrast

identifying morphology, size, and arrangement of a sample

3 broad types of staining

simple

differential

structural

types of dyes used in staining (for this class)

cationic

anionic

cationic dye

basic, positively charged chromophores

positive ion exhibits color

e.g. methylene blue, crystal violet

anionic dye

acidic, negatively charged chromophores

negative ion exhibits color

e.g. acid fuchsin, congo red, nigrosin

trick to remember anionic dyes

anionic dyes are acidic, negatively charged

negative stain

a stain that doesn’t penetrate the cell; background is colored

stain is repelled by the negative charge of the cell surface

usefulness of negative stains

good for examining morphology and size of cells (no heat fixation → no distortion of cells)

useful for organisms that don’t stain easily

example of a negative staining dye

nigrosin (a black anionic dye)

simple stain

a stain in which one dye is used to stain all the cells present

colors cell surfaces, not background

usefulness of simple stains

useful in distinguishing different morphologies, arrangement, relative sizes

provides contrast of colored cells when using bright field microscopy

example of a simple staining dye

crystal violet (a cationic dye)

If a smear is dry to the touch, can I just skip heat fixing?

No

Can I smear a sample from a slant or plate without water?

No

add 1 loopful of water to the slide first

Can I blow on a slide to dry a smear?

No, let it air dry

Which objective of the microscope should you use?

100x objective with immersion oil

How to store a microscope

make sure all immersion oil is cleaned off of the 100x objective using lens cleaner and a kimtech wipe

the stage should be lowered as much possible

4x objective should be moved into position

cord should be properly wrapped up

Why is immersion oil important for 100x microscopy?

Oil stops refraction of light between the specimen and the lens

(oil has the same refractive index as glass)

calculation fo resolution (resolving power)

d = wavelength / (2 x numerical aperture)

resolution = illumination (in nm) divided by light gathering capacity of the lens

cocci shape

round, spherical

bacilli shape

rods

vibrio shape

comma shaped

spirillum shape

spiral

pleomorphic shape

not one specific shape