Microscopy, Staining, and Microorganism Classification

Staining

Technique to enhance visibility of microorganisms.

Resolution

Ability to distinguish two close objects clearly.

Contrast

Difference in color or brightness between objects.

Fixation

Process of attaching specimens to slides.

Labeling

Identifying slides with initials, date, and content.

Dyes are used for

determining if negative or positive

Chromophore

Color component of a dye used in staining.

Acidic dye

Dye with chromophore in the negative ion.

Basic dye

Dye with chromophore in the positive ion. That’s what most bacteria have

Simple stain

Single basic dye used for cell observation. Crystal violet, safranin, methylene blue. shape, and arrangement of cells

Differential stain

Multiple dyes used to identify bacteria types.

Circle

coccus

Oval

bacillus

Spiral

spirilla

Two

diplo

Four

tetra

Cluster

staphylo

Chain

strepto

Gram stain

Common differential stain for bacteria classification.

Mordant

Substance that intensifies staining effect.

Decolorizer

Agent that removes color from certain cells.

Counterstain

Second stain used to visualize decolorized cells.

Gram-positive bacteria

Bacteria with thick peptidoglycan retaining purple color.

Gram-negative bacteria

Bacteria with thin peptidoglycan, appear pink after staining.

Acid-fast stain

Staining method for bacteria with waxy cell walls.

Taxonomy

Science of classification and naming of organisms.

Species

Group of organisms that can interbreed.

Strain

Variation within a species, especially in microbes.

Nucleic acid sequencing

Method for determining the sequence of nucleotides.

Biochemical tests

Experiments to identify microbial metabolic characteristics.

Special stains

Techniques for visualizing specific structures like endospores, flagella, and capsules

heating

kills and attaches bacteria to the slide

Preperation

·         Label your slide with sticker on the top. (Initials, date, what it is)

·         Draw a circle on the bottom the size of a dime.

·         If it is a liquid then don’t do this. If it is not a liquid then

put H2O droplet on top

·         Smear culture on thin film over slide

·         Let it air dry!

·  Pass the slide through flame to fix it (heat fix)

gram negative and positive testing

1.      Flood the slide with crystal violet for 1 minute, then rinse with water. (stained purple now)

2.      Flood slide with iodine for 1 minute, then rinse with water. (iodine acts as a mordant, all cells remain purple but darker)

3.      Slide is rinsed with solution of ethanol and acetone for 10-30 seconds, then rinse with water (smear is decolorized, gram positive cells remain purple, but gram negative cells are now colorless)

Slide is flooded with safranin for 1 minute, then rinsed with water and blotted dry ( gram-positive cells remain purple, gram negative cells are pink)

After crystal violet and iodine all cells will be  

dark purple

after alcohol, gram negative will be

colorless

after safranin, gram negative will be

pink

after safranin, gram positive will be

purple

acid fast stain steps

1. Carbolfuchsin (red) applied for 1 minute

2. Rinse with water

3. Wash slide with acid-alcohol

4. Rinse with water

5. Methylene blue applied for 1 minute