week 10 - isolating microbes

Question 1

why isolate microbes from the natural environment when we can sequence environmental DNA

Answer 1

• Is isolating pure cultures in the laboratory still needed despite sequencing
• We need to be able to isolate and grow microbes if we:
  o Want to use them
   To make some product for us (beer, antibiotics, enzymes, plastics, biofuels)
  o Want to study them:
   Genome sequencing is not enough
   We only know genes that have been studied in other microbes we have isolated before
  o Want to prove they cause a disease or cause a benefit (Koch’s postulates)

Question 2

what can we predict from genome sequence

Answer 2

o Metabolic network
o Antibiotic resistance genes
o But still >30% of genes are FUN – function unknown

Question 3

enriching for TNT degrading aerobic microbes

Answer 3

No other alternate energy source
* E.g. glucose

Need to add all the elements that microorganism need to grow without any other carbon sources

Need to give microorganisms time
* TNT not easy to degrade

Need to think about a suitable source of the microbes
* E.g. near where its developed?
* E.g. near explosion?

Microbes that use TNT grow more -> can transfer to the next
* Repeat to get enrichment culture
* Relative proportion increased

Question 4

enrichment and isolation: from nature to culture:
two steps:

Answer 4

1. Enrichment to increase abundance from passage to passage (several successive transfers usually needed)
2. Isolation of abundant organisms from last passage of enrichment culture by dilution to obtain pure culture
   - Twice at least to ensure it’s a pure culture

Question 5

ENRICHMENT

Answer 5

• Increases the abundance of those microbes with desired properties over time by selecting for those properties
  o Say degradation of TNT by using TNT as a growth substrate, converting TNT into biomass and CO2
• You take a sample from nature containing all sorts of microbes (e.g. TNT contaminated soil or just ‘normal’ soil) to inoculate a batch culture containing TNT
• Once the batch culture has grown (or TNT has been consumed), you dilute the culture into a fresh medium
• Over successive transfers, those microbes that can use TNT will increase in frequency
• The microbe that grows fastest under given conditions will become the most abundant
  Now isolate

Question 6

ISOLATION

Answer 6

• A colony formed from a single cell is a pure culture (a clone)
• Such an isolate is called a strain
• Obtain pure culture by dilution of mixed culture so you end up with single cells
  o A single cells in a microtitre well
  o A single cell far apart from other cells on an agar plate
• Do not use enrichment culture as a first step if you want to isolate those species which are most abundant in nature
  o In this case, directly dilute the environmental sample to single cells to obtain a pure culture

Question 7

EXAMPLE:
How to enrich a benzene degrading aerobic microorganism?

What is benzene

Answer 7

• Hydrocarbon
• Aromatic
• Act as a carbon source

Question 8

EXAMPLE:
How to enrich a benzene degrading aerobic microorganism?

what microorganisms

Answer 8

• Petrol stations
• Near an oil spill
• Seepage from waste disposal sites

Question 9

EXAMPLE:
How to enrich a benzene degrading aerobic microorganism?

isolate a batch culture

Answer 9

• Put in thing with benzene

Give it all the things it needs to grow (no carbon source so HAS to get energy from benzene)

Question 10

EXAMPLE:
How to enrich a benzene degrading aerobic microorganism?

what should the medium for the enrichment culture contain

Answer 10

• Benzene
• Sulphur
• Nitrogen (ammonium or nitrate better as a source)
• Oxygen
• Phosphate
• Iron
• water
  Along with oxygen (because it is aerobic)

Question 11

EXAMPLE:
How to enrich a benzene degrading aerobic microorganism?

what should the medium not contain

Answer 11

• Alternate carbon sources (glucose)
• Hydrogen in the form of H2 (should include it in the form of water)

Question 12

EXAMPLE:
How to enrich a benzene degrading aerobic microorganism?

growth conditions?

Answer 12

• Shake to provide oxygen
• Temp. similar to temp. where sample was taken from
• No light (don’t want phototrophic organisms)
• pH similar to where sample was taken

Question 13

EXAMPLE:
How to enrich a benzene degrading aerobic microorganism?

enriching

Answer 13

Then enrich –> increase abundance passage to passage (several successive transfers)
The microbe that grows the fastest will become the most abundant
Then isolate the abundant organisms from the last enrichment passage by dilution to get a pure culture