Enrichment & Isolation of Purple non-sulfur Bacteria


Cautions
Caution

As with most of the experiments in this course, you will be handling a variety of undomesticated organisms of unknown identity or pathogenicity. Handle all cultures with respect and using standard microbiological procedures.

Be extremely careful with syringe needles. These are sterile when first opened, but can inflict a painful wound if treated carelessly. Be especially careful with used needles, both because of their microbiological cargo and because of their lack of the initial dose of lubricant that makes them slide easily through the bung the first time used.

When removing plates from the anaerobe jars, keep them level at all times, and start by opening them and tapping any condensed water trapped in the lid out onto a paper towel.


Introduction

Purple non-sulfur Bacteria are anaerobic, facultative or obligate phototrophs. Enrichment of these organisms therefore relies on providing an anaerobic environment without a fermentable carbon source and plenty of light. The anerobic environment is generated microbiologically - aerobes use glycerol until the oxygen is depleted, and then are unable ferment it and so stop growing. Not many organisms can grow on glycerol anerobically, but photosynthetic anaerobes can grow either autotrophically (getting carbon from CO2) or photochemotrophically (using glycerol for carbon but not energy). The tubes turn dark brown to bright red, or sometimes green, because of the organisms photopigments.

If the tubes are incubated in the dark, or if the inoculum is very low in purple non-sulfur Bacteria, sulfate reducers such as Desulfovibrio can take over. These anaerobic organisms can grow heterotrophically on glycerol, not by fermentation but by using sulfate (instead of oxygen) as a terminal electron acceptor, generating sulfide. This results in the tube turning grey or black.

We also sometimes get Clostridium, some species of which can ferment gylcerol. These are rod-shaped endospore-formers, typically turn the tubes a pale sickly yellow-ish color, and form very flat colonies.

Patience in the key - these organisms are most often slow-growing. Because we aren't using strict anerobic methods (innoculating both liquid media & plates aerobically), this enrichment misses the more oxygen-sensitive species.


Materials

  • pond water
  • Balch bungs
  • Gaspack anaerobe jars and CO2/H2 generater packs
  • slides, coverslips, needles, syringes
  • Basic Isolation Media (BIM):

    Add to 1 liter of distilled water:

    • 1 gram (NH4)SO4
    • 0.5 gramK2HPO4
    • 0.2 gram MgSO4
    • 2 grams NaCl
    • 5 grams NaHCO3
    • 1.5 grams Yeast extract
    • 1.5 grams Glycerol

    Adjust pH to 7 w/ dilute HCl or NaOH

    For tubes, autoclave and dispense 20ml into each of about 50 sterile 25ml Balch tubes (use aluminum foil to cover the tubes for autoclaving and storage after the media is added)

    For plates, add 15g agar, mix & autoclave. Cool to about 50C & pour into plates


Procedure

  1. Add pond water to 20ml BIM in a 25ml Balch tube to the top of the neck of the tube so no air is left.
    Balch tube
  2. Put a syringe needle into a bung with the tip just through to the inside.
    Bung
  3. Put a paper towel over the base of the needle (to absorb the liquid displaced from the tube by the bung), then use it to stopper the bottle. The idea is to get the bung in trapping as little air in the tube as possible. The needle allows the displaced air (and fluid) to escape when you push the bung in, and allows the escape of gas generated during the incubation. If you can't press the bung in, it probably means the needle isn't quite far enough in. Leave the needle in place during incubation to allow gas to escape.
    Ready
  4. Incubate for 2-3 weeks in the 30C lighted incubator.
  5. Examine weekly for appearance, and make wet-mounts of samples drawn from the tube by syringe. Growth of purple non-sulfur bacteria is indicated by red, purple or brown color. Growth of sulfur reducing organisms is indicated by an opaque black color (metal sulfides).
    tubes
  6. When the tube is brightly colored , mix and draw a sample of the enrichment from the tube by syringe and streak onto a BIM plate. Seal plates in anaerobe jars with an active CO2/H2 generater and Paladium catalyst, and incubate as before for 1 week.
    Anaerobe jar
  7. Streak out a fresh plate with a well-isolated pigmented colony. Incubate 1 week as before. Examine individual colonies carefully and make wet mounts for microscopic morphology.
    coloniescolonies colonies
  8. Restreak until a pure culture is obtained. This can be very difficult; colonies are often small and difficult to isolate.

Observations

Can you identify the genus of your isolate? The purple non-sulfur Bacteria can be generally classified by gross microscopic morphology into 4 groups: Rhodomicrobium, Rhodobacter, Rhodopseudomonas and Rhodospirillum. Another group is Rhodocyclus, which looks like a lockwasher, but we have yet to isolate this specie. In previous semesters, the majority of isolates have been Rhodomicrobium, but Rhodospirillum is also common.

Rhodomicrobium

Rhodomicrobium
(stalked, often in chains)
Rhodobacter

Rhodobacter
(very large ovals)
Rhodopseudomonas

Rhodopseudomonas
(straight/bent rods)
Rhodospirillum

Rhodospirillum
(large spirals)