Chapter 3 Staining & Microscopy

Question 1

What does ‘Fix’ mean in staining? And what is the function?

Answer 1

Heat sample by Sliding with sample over flame 3 times quickly.

To adhere sample to slide. (But can kill sample)

Question 2

Why do we stain specimens?

Answer 2

To add color for better view and identify organism

Question 3

What are the reasons for using Basic stains?

Answer 3

Bacteria are negatively charged, so by using positively charged Basic stains, the stain will stick better to the sample

Opposite charges attract

Question 4

List some Basic stains.

Answer 4

methylene blue, malachite green, safranin red, crystal violet

Question 5

List some Acidic stains.

Answer 5

eosin red, India ink purple blue, nigrosine brown

Used primarily to color the Background

Question 6

What is negative staining, and what is the purpose?

Answer 6

uses an acidic stain to look for capsules

Question 7

What is the Simple staining technique used for?

Answer 7

Color background of specimen.
Used to identify shape, size and arrangement of bacteria

Question 8

What is the Differential staining technique used for? And how does it work?

Answer 8

Identify and distinguish between different organisms in a sample

Uses More than 1 dye of contrast colors

Question 9

What is the Gram Stain used to differentiate between?

Answer 9

Gram + and gram - organisms

Gram +: 40+ layers peptoglycan that make up cell wall
Gram-: 1 layer peptoglycan

Question 10

What is the Acid Fast Stain used to determine?
What colors should appear?

Answer 10

If lipid coat present outside cell wall

Lipid coat= Acid Fast= RED
No lipid coat= Non acid fast= Blue

Question 11

What is the Special staining technique used to identify?

Answer 11

Identify cell structures like flagella, endospores and capsules

Question 12

If I was to do a gram stain what steps do I follow?

Answer 12

• [ ] Stain both purple
  - [ ] Use iodine to bond with purple and hold in place
  - [ ] Alcohol used to remove outer membrane of gram - and purple color
  - [ ] Safranin (red) taken up by both cells but only shown in gram -

Question 13

Name the special stains

Answer 13

Negative stain
Endospore stain
Flagella stain

Question 14

What is the purpose of doing Negative Stain?

Answer 14

Color background with acidic (-) stain to identify capsules.

Example sentence: Negative stain is used to visualize the capsules surrounding certain bacteria.

Question 15

What is the purpose of doing Endospore Stain?

Answer 15

Endospore stain is used to identify bacteria that are capable of forming endospores.

Question 16

What is the purpose of doing Flagella Stain? Why is this important

Answer 16

Determine if flagella present

# and location of flagella can help determine organism.

Question 17

Explain the Shafer Folten stain

Answer 17

Form of identifying endospores

green is forced into the endospores by steaming the bacterial emulsion.

Question 18

Explain the Shafer Folten stain

Answer 18

Form of identifying endospores

green is forced into the endospores by steaming the bacterial emulsion.

Question 19

What is Mordant (iodine) used for?

Answer 19

Flagella stain- Coat flagella to become visible.

Gram stain - dye adhere better to specimen.

Question 20

Tell the lens power of each type of magnification:
Scan
Low
High
Oil immersion

Answer 20

Scan: 4X
Low: 10X
High: 40X
Oil immersion: 100X

Question 21

What is the equation to find total magnification?

Answer 21

Microscope power X 10 (oculars) = total magnification

Question 22

Light scope

Answer 22

Use long wavelength, gives poor resolution

Question 23

Dissecting scopes

Answer 23

Use short, wavelengths, better resolution, but worse magnification

Used when dissecting

Question 24

Describe Brightfield

Answer 24

Background= light
Specimen= dark

Question 25

Explain Darkfield

Answer 25

Background= dark Specimen = light

Question 26

When using darkfield microscopes, what is important to remember about staining?

Answer 26

Do not stain samples because you can kill the organism. Darkfield is used primarily with living organisms.

Question 27

Phase contrast Microscopy

Answer 27

Studies the internal organs of living organisms

Question 28

Fluorescence microscopy

Answer 28

Uses Short wave of light to reflect images back at longer wavelengths Flourochromes used to identify organisms

Question 29

What are floral chromes and why are they important?

Answer 29

Dyes Important for identifying certain organisms Certain florochromes are known to be attracted to specific organisms

Question 30

What form of microscopy is used to study physiology of a cell?

Answer 30

Confocal microscopy Gives 3-D clear images by using a laser light to reflect back images that are then put together by a computer

Question 31

Electron microscope microscopy

Answer 31

Only used to study dead organisms 2 types: TEM- electron beam trough sample. No 3D image SEM- scans the surface of the sample and gives a clear 3-D image of the surface only