Simple and Negative Staining

Question 1

What is the purpose of staining?

Answer 1

to increase contrast (highlights structures in biological tissues for viewing with the aid of a microscope

Question 2

What is a smear? What is the purpose of the heat fixed smear?

Answer 2

A smear is a thin film of a solution of microbes on a slide.

Purpose: it helps adhere and inactivate proteases (fixed to attach the microbes to the slide and to kill the microbes)

Question 3

What is aseptic technique?

Answer 3

a method designed to prevent contamination from microorganisms.

Question 4

Most common basic stain and its charge

Answer 4

Methylene blue (+)

Question 5

Most common acidic stain and its charge

Answer 5

Nigrosin (-)

Question 6

Why use a negative stain

Answer 6

Microbe is sensitive to heat fixation step

Question 7

Are most microbes positively or negatively charged?

Answer 7

Negative

Question 8

What does a negative stain do?

Answer 8

Stains the background rather than the cell

Question 9

Simple stain

Answer 9

Use of a single basic dye

Question 10

What does mordant do?

Answer 10

holds the stain or coats the specimen to enlarge it.

Question 11

What are three dyes used in simple staining?

Answer 11

1. methylene blue
2. crystal violet
3. safranin

Question 12

What dye is used for negative staining?

Answer 12

nigrosin

Question 13

What is the procedure for simple staining?

Answer 13

1. Create smear using aseptic technique: Flame inoculating loop, flame tube mouth, get sample, reflame tube mouth and replace cap, apply sample to slide and then reflame the inoculating loop.
2. Allow smear to dry and heat fix it by passing it over the flame a few times.
3. Place on tray and stain the smear with a simple stain such as methylene blue, crystal violet, or safranin and allow to sit for 30 seconds to a minute depending on the stain (i.e. methylene blue 1 min, crystal violet 30 sec.)
4. Rinse slide thoroughly with water and pat dry with kim wipes.
5. Place on microscope stage

Question 14

How does the procedure change with a slant as opposed to a broth?

Answer 14

With a broth you dip the inoculating loop into the broth and spread on the slide. With a slant you place a drop of water on the slide and then take a tiny pinhead sample of the bacterial growth and mix it into the water.

Question 15

What is the procedure for negative staining?

Answer 15

1. Place a small drop of nigrosin near one end of slide.
2. Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop.
3. Use another clean slide to spread the drop of stain containing the organism across the slide.
4. Allow the smear to dry without heating.
5. Place on microscope stage

Question 16

What are three organisms we looked at using simple stains? What dye did we use? What was the cellular mophology and arrangement? (what did we see?)

Answer 16

1. Staphylococcus epidermidis: methylene blue, blue clustered spheres (Gram positive; facultative anearobe; catalase-positive, coagulase- and oxidase-negative;
2. Bacillus cereus: methylene blue, blue single rods
3. Escherichia coli: methylene blue, blue rods in singles or paired

Question 17

What are three organisms we looked at using negative stains? What dye did we use? What was the cellular mophology and arrangement? (what did we see?)

Answer 17

1. Micrococcus luteus: nigrosin, black background with clear clustered spheres
2. Bacillus cereus: nigrosin, black background with single clear rods
3. Rhodospirillum rubrum: nigrosin, black background with single clear spirals

Question 18

What are the colored molecules in stains called and what part gives it its color?

Answer 18

chromogen

chromophore gives color

Question 19

What is the charged portion of a chromogen which allows it to act as a dye?

Answer 19

Auxochrome

Question 20

In a basic stain, what causes the cell to become colored?

Answer 20

The positively charged auxochrome is attracted to the negative charges on the surface of most bacterial cells.

Question 21

In a negative stain, what causes the background to stain and not the cell?

Answer 21

The chromogen is acidic and carries a negative charge. The negative charge on the bacterial surface repels the negatively charged chromogen so the cell remains unstained against a colored background.

Question 22

What information can be obtained from negative staining?

Answer 22

it is used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heat-fixing. It also produces minimal cell shrinkage so is useful when determining the accurate size is crucial.