Staining (microscopy)

Question 1

What is staining?

Answer 1

With coloured/ fluorescent chemicals that bind to cell organelles and makes them visible allows to identify different types of cells and different organelles (providing contrast)

Question 2

What are stains in microscopy?

Answer 2

Are used to increase the contrast of a sample by selectively binding to certain structures within the sample and changing their optical properties

Question 3

What is a contrast in biology?

Answer 3

Refers to the degree of visibility or differentiation between different structures or cells within a sample

Question 4

What would happen without staining?

Answer 4

Many structures within a sample may appear transparent or have similar refractive indices, making it difficult to distinguish between them

Question 5

How do stains help address this issue?

Answer 5

By making certain structures or cells more visible and thus increasing the contrast

Question 6

What are simple stains?

Answer 6

• they involve the use of a single dye to colour all of the cells or structures in a sample
• methylene blue can be used to stain DNA
• The stain is positively charged so attracted to the negatively charge DNA. All DNA will stain the same, from bacteria to humans

Question 7

What can you see in simple stains?

Answer 7

1) the shape
2) some basic structures
3) relative size

Question 8

What are differential stains?

Answer 8

Uses multiple dyes to distinguish different types of cells or structures based on their physical or chemical properties. The second dye is called a COUNTERSTAIN. Usually it’s a contrasting colour to the principal stain.

Question 9

What are differential stains used for?

Answer 9

• contrast two cell types
• indicate cell parts (eg gram stain and endospore stain)

Question 10

What are most stains in microbiology like and what does this involve?

Answer 10

• these involve the use of more than one dye (so that certain differences between cell type and structure can be distinguished)

Question 11

What is the most important differential stain?

Answer 11

The Gram Stain

Question 12

Why is staining used when preparing specimens to be examined under a microscope?

Answer 12

• so the cells and their contents are visible
• to increase contrast
• so organelles can be seen eg nucleus

Question 13

What are the two different gram stain groups?

Answer 13

Gram positive (violet) and Gram negative (red)

Question 14

What is the gram stain technique for?

Answer 14

It is used to separate bacteria into two groups, Gram positive bacteria and Gram negative bacteria

Question 15

How is Gram - positive tested?

Answer 15

• Crystal violet is first applied to a bacterial specimen on a slide
• then iodine, which fixes the slide
• the slide is then washed with alcohol

-the gram positive bacteria retains the crystal violet stain and will appear blue/purple under the microscope

Question 16

How are gram negative tested?

Answer 16

They have thinner cell walls, and therefore they lose the stain.

They are then stained with safranin dye, which is called a counterstain

These bacteria will appear red

Question 17

How are gram positive bacteria when it comes to penicillin?

Answer 17

They are susceptible to the antibiotic penicillin, which inhibits the formation of cell walls

Question 18

How is gram negative bacteria with penicillin?

Answer 18

They have much thinner cell walls that are not susceptible to penicillin

Question 19

What is the Acid-Fast technique used for?

Answer 19

To differentiate species of Mycobacterium from other bacteria

Question 20

Explain the Acid-Fast technique

Answer 20

A lipid solvent is used to carry carbolfuchsin dye into the cells being studied

The cells are then washed with the dilute acid-alcohol and retain the carbolfuchsin stain, which is bright red

Other bacteria lose the stain and are exposed to a methylene blue stain, which is blue

Question 21

Explain the 4 stages involved in the production of these slides.

Answer 21

• FIXING : chemicals like formaldehyde are used to preserve specimens in as near-natural state as possible
• SECTIONING : specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. this can then be sliced thinly with a knife called a microtome
• STAINING : specimen are often treated with multiple stains to show different structures
• MOUNTING : the specimens are then secured to a microscope slide and a cover slip placed on top

Question 22

Explain the Risk Management

Answer 22

• many stains are toxic / irritants.
• risk assessment must be carried out before any practical work is started (to identify specific procedures that can cause harm)

Question 23

What is CLEADSS?

Answer 23

Provide student safety sheets that identify specific risks, advise on the measures to be taken to reduce these risks and the action to be taken in any emergencies

Question 24

SUGGEST WHY GRAM-NEGATIVE INFECTIONS ARE MORE DIFFICULT TO TREAT THAN GRAM-POSITIVE INFECTIONS

Answer 24

• gram negative have a thinner wall
• penicillin disrupts cell wall
• less cell wall formation (in gram negative)
• membrane (around gram negative) prevents entry of penicillin

Question 25

DESCRIBE PRECAUTIONS WHEN USING CRYSTAL VIOLET & POTASSIUM IODIDE (CLASSED AS IRRITANTS)

Answer 25

- avoid skin / eye contact - wear gloves / goggles

Question 26

In order to investigate onion cells, a stained temporary mount was made. Suggest what a good stain to use.

Answer 26

Iodine or methylene blue

Question 27

Why did the onion cells need to be stained?

Answer 27

To observe the cells, nuclei and cell walls absorb the stain more strongly, so that they can be distinguished

Question 28

What is the advantage of making a temporary mount of cells?

Answer 28

Temporary mounts are quick to prepare; the cells can be observed whilst alive

Question 29

A scientist wishes to study yeast cells using a light microscope. How should the slide be prepared?

Answer 29

To create a temporary mount, yeast cells should be placed on a slide with a droplet of iodine solution should be added to stain the organelles in the cell. A cover slip should be placed over the cell. A squash slide can be prepared by gently pressing down on the cover slip.

Question 30

Stains are used for a number of different purposes when preparing slides. Give 3 examples of how they can be used.

Answer 30

Increase contrast, so different parts of a cell can be distinguished. to observe the location of a certain chemical in a cell Differentiate between organisms that can be hard to tell apart