Staining (microscopy) Flashcards

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1
Q

What is staining?

A

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)

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2
Q

What are stains in microscopy?

A

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

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3
Q

What is a contrast in biology?

A

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

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4
Q

What would happen without staining?

A

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

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5
Q

How do stains help address this issue?

A

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

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6
Q

What are simple stains?

A
  • 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
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7
Q

What can you see in simple stains?

A

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

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8
Q

What are differential stains?

A

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.

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9
Q

What are differential stains used for?

A
  • contrast two cell types
  • indicate cell parts (eg gram stain and endospore stain)
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10
Q

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

A
  • these involve the use of more than one dye (so that certain differences between cell type and structure can be distinguished)
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11
Q

What is the most important differential stain?

A

The Gram Stain

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12
Q

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

A
  • so the cells and their contents are visible
  • to increase contrast
  • so organelles can be seen eg nucleus
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13
Q

What are the two different gram stain groups?

A

Gram positive (violet) and Gram negative (red)

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14
Q

What is the gram stain technique for?

A

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

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15
Q

How is Gram - positive tested?

A
  • 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

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16
Q

How are gram negative tested?

A

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

17
Q

How are gram positive bacteria when it comes to penicillin?

A

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

18
Q

How is gram negative bacteria with penicillin?

A

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

19
Q

What is the Acid-Fast technique used for?

A

To differentiate species of Mycobacterium from other bacteria

20
Q

Explain the Acid-Fast technique

A

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

21
Q

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

A
  • 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
22
Q

Explain the Risk Management

A
  • 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)
23
Q

What is CLEADSS?

A

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

24
Q

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

A
  • 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
25
Q

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

A
  • avoid skin / eye contact
  • wear gloves / goggles
26
Q

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

A

Iodine or methylene blue

27
Q

Why did the onion cells need to be stained?

A

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

28
Q

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

A

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

29
Q

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

A

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.

30
Q

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

A

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