Lecture 3

Question 1

Gram-negative vs Gram-positive

Answer 1

Gram positive: has a thick cell wall made of peptidoglycan

Gram negative: has an outer membrane that is also a bilayer, but is made up of LPS.
- each species has a very unique LPS

Question 2

How to interpret Gram stain results?

Answer 2

purple: Gram positive
pink: Gram negative

Question 3

what is special about Archaea membrane?

Answer 3

• they have an S-layer (slayers)

which are layers of protein that make a crystalline structure (forms patterns).

Question 4

when are pure-culture studies used?

Answer 4

• You can use one for a couple of species
  
  • Use synthetic media
  • If you want to prove an organism is proving something from the environment, you can grow the bacteria on a specific medium and graph the growth rate (how fast that this nutrient is being broken down)

Question 5

when are microcosm studies used? / what is microcosm

Answer 5

Microcosm:
- You go to the environment, isolate something from the environment, put it into the system (for example Winogradsky column)
- The Winogradsky column allows us to enrich for sulfate reducing bacteria.
- This would feed purple bacteria.

Winogradsky:
Top layer: aerobic
Bottom layer: gets more and more anaerobic

The microorganisms that grow in the top layer (aerobic) will not grow in the bottom layer. This mimics environmental conditions, and we can study a variety of microorganisms that have different growing conditions all within the same column.

** essentially a natural habitat that is mimicked in the lab. Good for microbes that are non-culturable in pure culture in the lab.

Question 6

what is culture independent (in situ) studies?

Answer 6

• You don’t manipulate the environmental sample
  
  • Take the environmental sample and directly analyse it
  • Put little collectors on the soil and measure the gases on the soil
    ○ To not disturb the environment
    ○ No manipulation, at all.
    ○ In situ (on site).

Question 7

what is FISH? how to tag a gene?

Answer 7

FISH
- Fluorescent in situ hybridization.
- Take a DNA probe linked to a fluorescent tag
- The fluorescent tag will give a wavelength of fluorescence (different colours)
- DNA tag matches specific part of 16s rRNA sequence.
Tagging a gene
- Stick cells on microscope slide to kill them
- Add chemicals to denature DNA
- Ds –> ss
- Add the probe and it will bind to the ssDNA.
- If not the right species, the label won’t stick
○ Will only stick to very specific organisms.

Question 8

what is qRT-PCR (quantitative real-time PCR)

Answer 8

• PCR heats, denatures, cools down , amplifies
  
  • Each cycle the DNA product is logarithmically growing
  • You will reach a certain plateau.
  • After 30 cycles of heating a cooling: you are at 2 Fluorescent units
    ○ Units represent how much product is made
    ○ qRT-PCR:
    § Fluorescence increases as amount of DNA increases
    § OR
    § Fluorescence decreases as amount of DNA increases
    ○ qRT-PCR machine heats, cools and ALSO has a little UV source for every well.
    If there are 96 wells, you can measure the fluorescence of all 96 wells at the same time with little probes in each well in a dilution plate, for example.