Microscopy

Question 1

What determines the smallest object we can see?

Answer 1

• size at which objects become visible depends on resolution of observers eye

*res = smallest distance between 2 objects that allows us to see them as seperate objects

• resolution determined by the distance between 2 foveal pixels (group of cones with neurons in the portion of the retins where photoreceptors are packed)
• we can detect a group of smaller items when in a group but cannot resolve them as individuals

Question 2

describe magnification

Answer 2

• need to magnify to resolve (inc the objects apparent size/dimension
• detection = ability to determine the presence of an object

*eukaryotes range from 10-100 um, prok range from 0.4-10 um

Question 3

examples of size based contradictions of microbes

Answer 3

• some can be seen with the naked eye: Thiomargarita namibiensis
• some function as cell communities ex: biofilms (a whole community of bacteria either 1 species or several, we need to study as community)
• some microbial bacterial communities should be studies as whole entities: the human gut/microbiome
• some viruses are as large as bacteria: mimivirus, mamvirus

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Question 4

what are the different instruments to look at microbes

Answer 4

• light microscopy: resolves images according to absorption of light
• Electron microscopy: uses means of electrons to resolve smaller details (smaller then the wavelength of visible light, can resolve viruses)

*electron beams have smaller wavelength so can resolve smaller things

• atomic force microscopy: uses intermolecular force to map 3D topography of the cell
• X-ray crystalography: detects the interference patters of X-rays entering the crystal lattice of a molecule

Question 5

what is the range of visible light? what is the conditions required for electromagnetic radiation to exist?

Answer 5

• visible light (400-700nm) is part of electromagnetic radiation (electrical and magnetic waves perpendiular to each other
• for electromag radiation to resolve an object the following must exist:
• wavelength of radiation much be equal or smaller than the size of the object (allows you to see that object as a seperate entity)
• there needs to be contrast between the object and its medium/background
• a detector with sufficient resolution for the given wavelength

*otpimal for 0.4-0.7 micro meters

Question 6

what are the 4 fates when light interacts with an object

Answer 6

1. Absorption: photons energy is aquired by the absorbing object

*use for birght feild microscopy (object appears dark), in dark feild the object is very bright

2. Reflection: the wavefront bounces off the surface of an object
3. Bending of light as it enters a substance that slows it speed
4. Scattering: the wavefront interacts with an object smaller than the wavelength of light

Question 7

what are the 4 types of light microscopy

Answer 7

4 types:

Bright field microscopy

Dark field microscopy

Phase-contrast microscopy

Fluorescence microscopy

Question 8

describe bright feild microscopy

Answer 8

• most common

*subcellular structures too small to resolve by light microscopy

• object appears as a dark silhouette
• resolution limit = 0.4-0.7 but magnification 1000x (greatest magnification that can imporve our preception of detail)
• greater magnification inc the image size but not the resolutions (empty magnification)
• can use oil emerson lens/microscopy

Question 9

what is oil emersion lens microscopy

Answer 9

• use in birghtfeild
• put a drop of oil (similar refractive index to glass lens) between the lens and the object minimizes loss of refracted light at the widest angles and sharpens image (see Fig 2.14)

Question 10

what is a compound microscope

Answer 10

• workhorse light microscope in gneral microbio labs
• has a system of multiple lenses designed to focus, correct &/or compensate for abberration

- ocular lens (10x magnification), objective elns (10x-400x mag) *needs to be parfocal)

• total magnification = magnification of the ocular multiplied by that of the objective

Question 11

what is a simple way to observe microbes

Answer 11

• place them in a drop of water on a slide with a coverslip
• called wet mount prep

Advatages: observation of cells in natural state

Disadvantages: little contrast between cell and background (want to stain cells), sample may dry out qucik (use mounting reagents)

*we are using chemicals which are quite harsh (alc and acids), this kills the cell and we

*detection and resolution under a microscope are enhanced by staining, staining does NOT improve resolution

Question 12

what is the difference between simple stain and differential stain

Answer 12

Simple stain

• adds dark colour specifically to cells, but not to external medium or surrounding tissue
• methylene blue is the most commonly used stain

Differential stain

• stains one kind of cell but not another
• most famous differential stain is gram stain

Question 13

Describe the staining procedure for Methylene Blue

Answer 13

• methylene blue is a general stain for cells, just adds colour
  1. put sample on slide with water so you can spread the sample
  2. allow to air dry which may shrink the cells
  3. add methanol to fix cells to the slide, allow to air dry
  4. stain with methylene blue (1 min)
  5. wash off stain with water

6, blot off excess water

7. view under microscope

Question 14

what is a differential stain, give an example and how it works

Answer 14

• stains one kind of cell but not another
• Gram stain differentiates between 2 types of bacteria
• Gram positive: retains the crystal violet stain because of thicker cell wall (cells appear purple)
• Gram negative: do not retain stain, cells appear pinkish/reddish

Question 15

explain the Gram stain procedure

Answer 15

1. Add methanol to fix cells to surface
2. Add crystal violet stain
   * stains gram pos cells reversibly
3. Add iodine which binds stain to Gram-pos cells
   * only gram pos cells have iodine complex with crystal violet to retain the stain
4. Wash with ethanol
   * stain is removed from gram neg but remains in gram pos
5. Add safranin counterstain
   * counter stains gram neg pink, gram pos stay purple

Question 16

explain the staining mechanism

Answer 16

• gram positive has multiple peptidoglycan layer
• gram neg have a thin peptidoclygan layer sandwhiched between two membranes
• when crystal violet it added it penetrated into the cell wall/ peptidoglycan (dye penetrates through all layes in gram pos)
• wash to remove excess
• by adding iodine, it complexes with the crystal violet and gets traped in peptidoglycan layers
• add the counter stain to stain cells that did not retain a lot of crystal violet

Question 17

what is a spore stain?

Answer 17

• another differential stain
• uses malachite green, detects endospores of Bacillus and Clostridium bacteria
• retained by spores bc very different cell walls

cells highlighted as unstained halos

Question 18

what is capsule staining

Answer 18

• also called negative staining
• its a differential stain
• does not stain the capsule, the halo you see is the spore around the bacteria cell
• stain interacts with background and cells highlighted as unstained halos

Question 19

what is dark field microscopy?

Answer 19

• dark feild optics enables microbes to be visualized as halos of bright light against darkness

*visualize live samples bc no fixation or staining required

mechansim: object scatters light and is collected by objective lens, light that just passes through slide shines outside the lens so backgorund is dark

*important for cells that are very thin

*see outline of cels but not detail

Question 20

What is phase-constrast Microscopy?

Answer 20

• PCM
• expolits differences in refractive index between the cytoplasm and the surrounding medium or between diff organelles
• reveals differences in refractive index as patters of light and dark

*can be used to view live unfixed organelles, useful for eukaryotes cells protozoa and ameba

* in picture the differences in refractive index reveals the nucleus, oral groove and cilia

Question 21

what is fluorescence microscopy

Answer 21

• powerful tool for detective ‘parts’ of cells
• specimen absorbs light as a defined wavelength and emits light of a longer wavelength plus some heat (fluorescence)
• scope fitted with light detectors, use different probes to detect components within cell after they have been labelled with a prob
• can visualize live (or fixed) cells in three dimensions

Question 22

What is Autofluorescence?

Answer 22

some cell components naturally fluoresce under specific light wavelengths (no stain required)

* will fluoresce when light of a certain wavelength is shined

• cells with chlorophyl will autofluorece
  ex: cyano bacteria

* in image the green is heterocyst which has been stained with a specific probe, the cells containing chlorophyl are autoflurorescing as red

Question 23

what are fluorophores?

Answer 23

• tags that are attached to a probe that interact with specific components of a cell

*fluorescent chemical compound with specificity for cellular target

• this specificity is determined by: chemical affinity, labelled antibody, DNA hybridization, Gene fusion reporter tags (when rpotein is expressed you see where is it located in cell)

Question 24

what is GFP

Answer 24

•Jelly fish Green Fluorescent protein (GFP)

• the DNA sqeuences coding for GFP can be spliced to that of a target protein gene sequence (to create a recombinant gene)
• this is then expressed into a protein (which is fluorescent bc of the GFP-tag)

* tag a specific component and you see that, you can also tag multiple components

Question 25

what is confocal laser scanning microscopy?

Answer 25

- form of advanced fluorescence microscopy where the sample can be scanned by laser light to reveal 3D topography - can use to view live cells - if looking at bacteria biofilm (bacteria growing atached to substrate) simple microscopy only sows whats ont he surface, if you want to see the whole community expose to diff fluorophoes that target diff ocmponents/metabolic acivity can get idea of whats going on (live and dead, or what is actively metabolizing) view with the laser scanning \* in picture live cells = green, dead = red

Question 26

What is electron microscopy?

Answer 26

- electrons are focused using magnetic feild/lenses to gnerate images of cells (and thier intracellular organelles/macromolecular complexes) - uses electron beam source, then focused in a field, sample is mounted on copper grid and uses a magnetic lens - resolution 100x more than lihgt microscopy - electrons behave in light waves but have high freq (generate heat) and allow resolution of robjects a few nanometers in diameter (0.0037nm wavelength)

Question 27

why cant EM be used to view live cells?

Answer 27

- the magnetic lenses (used to focus electron beams) and the sample are in a **vacuum** - cooled ot sub 0 temps and smaple often coated in heavy metal stains

Question 28

what are the two major types of electron microscopy?

Answer 28

**TEM: Transmission Electron microscopy** * Specimen is fixed and sliced into thin sections using a microtome. (cells added to matrix into block then that is sliced) * Electrons pass through the specimen and can reveal internal structures * Samples are supported on a copper grid (sample holder) **SEM: Scanning electron microscopy** * Electrons scan the surface of the (unsliced) specimen * Reveal external features/contours in 3-Dimension * Samples are supported on a copper grid (sample holder)

Question 29

descrcibe Cryo-electron microscopy

Answer 29

\*looking at diff in refractive index of different components - high strength electron beans permit low temp imaging **without** specimen staining - specimen must be **flash frozen t maintain cell in natural state** - water does not crystalize under these conditions, but forms a **glass amorphous phase** \* sample can be imaged without drying - useful for imaging very fragile cells, organelles, macromolecular complexes and microbial structures - Pros: high res in normal/native state (no chems tains or fixation),

Question 30

What is Cryo-electron tomography?

Answer 30

- 3D image construction - combines cryo-electron-EM with the ability to take high resolution images at different angles or with different focal planes - images are combined through a mathematical transformation to model entire object in 3D

Question 31

what is atomic force microscopy

Answer 31

- form of scanning probe microscopy - can be applied to living cells supended in water - tiny sharp tip attached to a **cantilever** measures the **van der waals forces** between electron shells of adjacent atoms on the cell surface and the surfect of the tip - sue to create image of a cell - enables nanoscale observations of cell surfaces