isolation

Question 1

liquid media
what are the 2 types?

Answer 1

liquid media are used to grow planktonic cells that don’t tend to be attached to one another (unless a truly filamentous organism).
Two key
types:
* defined media that use contain precisely known amounts of substances that have a known molecular weight only e.g. potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium chloride, magnesium sulfate,
ferric chloride, D-glucose, thiosulfate etc.

• complex media that contain anything you can’t define the molecular weight of, e.g. yeast extract, beef extract, peptone, tryptone, amylose, amylopectin etc.

Question 2

solid media
what about for high temperatures?

Answer 2

• solid media (same as liquid media but set with agar to make a gel) are used to grow colonies (piles of cells each derived from a single original cell – a clonal population). Often (but not always), these are biofilms
  (cells attached to a surface and to other cells).
• other gelling agents are used – some for high-temperature cultivation e.g. PhytaGel, silica gel, but often costly or technically annoying to work with.
• usually poured as plates (for isolation/purity checking) or slants (for maintaining cultures).

Question 3

Cell cultivation: broth culture (bottle types)

Answer 3

• Erlenmeyer flasks (250-mL normally, used for 50-mL
  culture) can be rapidly shaken to increase gas exchange and
  maintain strong growth. Used for growth curves and other
  times you want oxygen not to be limiting. Stopped with
  cotton wool or foam to let air in/gases out. Stopped with a
  vaccine stopper (e.g. SubaSeal) that can be injected through
  if growing under a non-air gas.
• universal bottles (c.30-mL) – hold about 10 mL culture and
  are used mainly for testing properties of an organism.
• MacCartney bottles (ditto) also used, as are Bijou tubes
  which are about 4-mL and good for making tiny agar slants.
• serum bottles are gas-tight if sealed with a vaccine stopper
  – can be used to grow anaerobes, methanotrophs etc that
  need non-air gas but in small volumes.

Question 4

Enrichment culture approaches

Answer 4

• elective enrichment:
  do something that enables an organism/functional guild to grow
  e.g. media containing only one C source, which only restricted groups can grow on (methane for methanotrophs, methanol for methylotrophs, benzoate for Pseudomonas spp., CO2 for autotrophs…)
  e.g. using an N-source only a limited group can use e.g. N2 gas for diazotrophs
• selective enrichment
  do something that prevents or minimises growth of certain groups
  e.g. adding a growth inhibitor such as cycloheximide to prevent growth of the Fungi
  e.g. lowering/raising the pH/temperature etc to limit growth to only specific groups
  e.g. Pasteurising media to kill vegetative cells so that only endospore-formers remain

Question 5

General sequence

Answer 5

1. inoculate 50-mL sterile broth in a 250-mL Erlenmeyer flask.
   incubate appropriately until growth evident
   2.subculture – remove 10 % of volume (5 mL) into 45 mL fresh broth in new flask
   incubate appropriately until growth evident
   3.subculture – remove 10 % of volume (5 mL) into 45 mL fresh broth in new flask
   repeat for usually 5-10 rounds of serial subculture
2. after serial subculture – plate out - either by streak plating or by serial dilution in 0.9 % w/v NaCl and spread-plating

Question 6

What are in defined media?

Answer 6

example – E-basal salts (per litre) – note no C/E source by default and N source can easily be omitted
* 4 g potassium dihydrogen phosphate (29.4 mM)
* 4 g dipotassium hydrogen phosphate (23.0 mM)
* 0.4 g ammonium chloride (7.5 mM)
* 0.8 g magnesium sulfate heptahydrate (3.2 mM)
* 10 mL Kelly’s solution “T” trace metals
[contains Zn(II), Ca(II), Mn(II), Co(II), Mo7O246-, Fe(II) and Cu(II) – more elaborate versions exist e.g. to isolate some methanogenic Archaea you need to add Ni(II) and orthotungstate (WO42-)]

• generally for soluble C sources, you’d aim for about 50 mM C so e.g. so divide amount of carbons by 50

Question 7

• isolate a generalist aerobic heterotroph that is a thermophile

Answer 7

• obtain a suitable inoculum – compost heap, coal spoil, leaf litter…
• prepare it – easiest to shake a small volume in 0.9 % w/v NaCl to liberate cells but leave most of the organic material behind
• use it to inoculate nutrient broth – incubate with shaking under air at e.g. 60 °C
• perform serial subculture, plating etc – use nutrient agar (may melt at 60 °C so using something like PhytaGel or silica gel instead of agar may be needed)

Question 8

• isolate a nitrogen-fixing heterotroph

Answer 8

• obtain a suitable inoculum – river or pond water, soil…
• prepare it if needed for solid material – easiest to shake a small volume in 0.9 % w/v NaCl to liberate cells but leave most of the organic material behind.
  For aquatic systems, passing many litres though a 0.22 µm pore-size membrane will trap Bacteria – you then use the membrane as the inoculum
• use it to inoculate a defined medium that contains no nitrogen sources and uses e.g. 10 mM D-(+)-glucose as the C-source – you would probably add Fe(II) and either Mo7O24
  6- or VO3 - depending on which nitrogenase you want
  to try and enable the use of.
• incubate with shaking under air at e.g. 30 °C
• perform serial subculture, plating etc – use the same defined medium with 10 mM D-(+)-glucose as the C-source

Question 9

isolate an endospore-forming organism than can grow on
paracetamol

Answer 9

• obtain a suitable inoculum – activated sludge, soil
• prepare it if needed for solid material – easiest to shake a small volume in 0.9 % w/v NaCl to liberate cells but leave most of the organic material behind.
• Pasteurise for 10 min at 90 °C to kill the majority of vegetative cells, leaving only endospores behind
• use it to inoculate a defined medium that contains 6.25 mM paracetamol as the only C-source – endospores will germinate and if they can use paracetamol as a C-source, grow.
• incubate with shaking under air at e.g. 30 °C
• perform serial subculture, plating etc – use the same defined medium with 6.25 mM paracetamol

Question 10

• isolate a methanotroph that uses pMMO

Answer 10

• obtain a suitable inoculum – activated sludge, soil, river water etc
• prepare it per previous slides
• use it to inoculate a defined medium that contains 1 µM cupric sulfate as this is needed for pMMO expression incubate with shaking under air supplemented with 20 % v/v methane and 10 % v/v CO2 at e.g. 30 °C
• perform serial subculture, plating etc – use the same defined medium with the same Cu content and the same headspace gases

Question 11

• isolate a chemolithoautotroph that uses tetrathionate (S4O62-) as the electron donor and nitrate (NO3-) as the terminal electron acceptor

Answer 11

• obtain a suitable inoculum – activated sludge, soil, river water etc
• prepare it per previous slides
• use it to inoculate a defined medium that contains 10-20mM potassium tetrathionate and 30 mM potassium nitrate in 30-mL Universal bottles filled to the brim and tightly sealed to exclude air
• perform serial subculture, plating etc – use the same defined medium with the same supplements and incubate under argon