Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Marine Microbial Ecology and Biotechnology > Lecture 5 Methods in Microbial Ecology and Biotechnology from cell counting to nucleic acids based methods > Flashcards

Lecture 5 Methods in Microbial Ecology and Biotechnology from cell counting to nucleic acids based methods Flashcards

1
Q

What did Antoni van Leeuwenhoek discover?

A

Discovered bacteria and is now called the father of microbiology

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is Kochs postulates?

A
  1. The suspected pathogen must be present in all cases of disease and absent from healthy animals
  2. The pathogen must be grown in pure culture
  3. Cells from a pure culture of the pathogen must cause disease in a healthy animal
  4. The pathogen must be reisolated and shown to be the same as the original
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is the Winogradsky column? And what can it show?

A

Organic matter is incubated with light and supplements and an environmental gradient is created, oxygen decreases and sulphide increases. This gradient creates specific niches for bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is standard plate counting? How do you work it out?

A

A method for counting microbial abundance. To work it out (=total number of colonies x dilution factor)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How many colonies should you have on a standard plate counts?

A

30-300

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is the colony counting technique for marine samples and what is its equation?

A

Membrane filter techniques

CFU/ml = CFU x dilution factor/volume

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is light microscopy used for and how can you help improve them?

A

See how many microbes are present directly, very useful as you dont have to grow them, but a stain can help improve contrast and differentiate properties

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the limit of compound microbes in light microscopy?

A

That a magnification of 1000x is required to resolve objects of 0.2μm, which is the size of the smallest microbes so cannot differentiate their properties

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How does fluorescence microscopy work?

A
  • Cells are made to fluoresce by illuminating them from above with light of a single colour
  • Filters are used so that only fluorescent light is seen, thus, cells appear to glow in a dark background
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Why is fluorescence microscopy considered easier than light microscopy?

A

Because it has a better contrast to visualize the bacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Name three benefits of fluorescence microscopy

A
  1. Use of natural fluorescence within cells
  2. Use of compound specific stains
  3. Enable in situ cell counts of individual cells instead of colonies, and independent of culturability
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Give two reasons why electron microscopes are good?

A
  • Highest resolution
  • Highest magnification
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What can a transmission electron microscope do?

A

See through cells to review intracellular structures

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What can scanning electron microscopes do?

A

Examine surfaces

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is the fastest way to count cells?

A

Flow cytometrey

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are the three steps of flow cytometry?

A
  1. The individual cells flow through a narrow tube
  2. Lasers shine through and a detector detects natural optical properties
  3. It also detects laser-stimulated fluorescence
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Why is flow cytometry so good in comparison to epifluorescence microscopy?

A

It has a much higher throughput

18
Q

What does fluorescence in situ hybridisation (FISH) allow for?

A

Allows for the visualisation and quantification of different microbes

19
Q

How is SSU rRNA used in FISH?

A

Used to pick a stretch unique to the organisms and attach a fluorescent dye fixed on a probe, which goes through the cell membrane

20
Q

How does FISH work?

A
  1. A fluorochrome is attached to specific DNA
  2. A specific temperature is used to combine the complementary bases so they fluorescent labelled oligonucleotides hybridize
  3. The excess probes are washed
  4. Then a microscope or flow cytometer is used to identify the microbes
21
Q

How does CAtalysed Reporter Deposition (CARD-FISH) differ to FISH?

A

Horseradish peroxidase probes (HRP) are used instead of the fluorochrome probes

22
Q

What are the two steps of CARD-FISH?

A
  1. HRP is hybridized with 16S rRNA
  2. Fluorescent tyramide is added and multiple molecules are activated by a single HRP, giving a much stronger fluorescence signal
23
Q

What is the advantage of CARD-FISH?

A
  • Actively growing cells with many rRNA signals present
  • Further amplification
  • Allows for the detection of small or even rare microbes within a mixed community
24
Q

Where are these from?

A

FISH or CARD-FISH

25
Q

What is a confocal Laser Scanning Microscope (CLSM)

A

A computer-controlled microscope that couples a laser to a fluorescence microscope

26
Q

How does CLSM work?

A

A laser beam is adjusted such that only a particular layer within a specimen is in focus at a time, creating a high contrast 3D image generated from the combination of several planes

27
Q

When are CLSM useful?

A

Microbial biofilms

28
Q

Where are these images from?

A

CLSM

29
Q

Name 6 ways you can detect microbial activities

A
  1. Microsensors
  2. Microautoradiography
  3. Stable isotope probing
  4. NanoSIMS
  5. Raman
  6. Gene expression - transcriptomics and proteomics
30
Q

What is a microsensor?

A

A small device that can pick up environmental changes, for example, the sulphur oxidation by cable bacteria

31
Q

What is microautoradiography?

A

An incubation experiments with radioactive labelled compounds (normally C14) to assess uptake activity to help distinguish processes

31
Q

What is microautoradiography?

A

An incubation experiments with radioactive labelled compounds (normally C14) to assess uptake activity to help distinguish processes

32
Q

What is Stable Isotope Probing (SIP)?

A

In situ incubation with stable-isotope-labelled compounds such as C13, which are incorperated in the DNA and a CsCL gradient separates DNA with heavy from light isotopes

33
Q

What is SIP useful for?

A

Linking microbial identity to a particular function

34
Q

What is NanoSIMS?

A

An imaging system that can examine up to 7 masses at a time

35
Q

How does the NanoSIMS work?

A

The NanoSIMS uses an ion source to produce a primary beam of ions. These primary ions erode the sample surface and produce atomic collisions, some of these collisions result in the release of secondary ion particles. These ions are transmitted through a mass spectrometer, where the masses are measured and identified

36
Q

How can FISH and NanoSIMS be used together?

A

To see what organism is responsible for the optical properties, as the dye in FISH can be used as a signal.

You can find out:

  • Where cells are
  • What they do
  • Fe take up
  • C take up
37
Q

Using FISH and NanoSIMS, UCYN-A produced this image and their genome lacks photosystem 2 and the Calvin cycle, what does this suggest?

A

That they have an ectosymbiotic relationship with a phytoplankton

38
Q

How does Raman-FISH work?

A
  • Raman microspectroscopy can differentiate raman shifts of heavy isotopes from light isotopes
  • It can be combined with FISH for cell identity
39
Q

How many microbes have been brought into culture?

A

Only 1%

40
Q

Give two reasons why we cannot culture more microbes

A
  1. Lack of knowledge of their growth conditions, the marine setting is normally oligiotrophic
  2. They may have commensal relationships and cannot live in isolation

Marine Microbial Ecology and Biotechnology (11 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions