Enrichment & Isolation of Polysaccharide Degraders


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.


Introduction

In some instances, enrichments are designed to obtain organisms with desired properties, regardless of taxonomic group; for example species than can degrade environmental contaminants or waste products. Some polysaccharides, such as chitin (from arthropods & fungi) and agar (from brown algae), are very difficult to degrade. Decomposition of these difficult polysaccharides is usually accomplished by fungi and members of the Cytophagales, a large group of common but poorly understood Bacteria.

In this enrichment, we rely on a simple artificial sea water to provide basic mineral/ion requirements, and add a polysaccharide (we're using agar) as the only carbon and energy source. The appropriate polysacchride degraders will, eventually, begin to break down these substrates as they grow. However, other organisms can also then grow from the sugars released by this degradation. Separation of the degraders from the organisms living on leftovers occurs when you plate the organisms out and force them to make a living by themselves - only the degraders can make a living as a colony on the plate unless the plate is overloaded with colonies.

Most often, we get one of two classes of bacteria in this enrichment; white colonies of the family Cytophaga and yellow colonies of the family Flavobacteria.


Materials

  • Enrichment (E) media = used artificial sea water (Instant Ocean).
  • Enrichment media plates = used artificial seawater with 15g agar/liter
  • Granulated agar
  • samples of aquarium sand, soil, sea sand, freshwater sediment, &c

Procedure

  1. Add a pinch of granulated agar to a culture tube containing 10ml of E media.
    tube
  2. Add a pinch or loop full of innoculum (marine or aquarium sand is best) to the tube of enrichment medium.
  3. Incubate at 30C. Examine microscopically for growth weekly (look especially for organisms stuck to bits of substrate).
  4. Streak a sample from the enrichment onto an E-agar plate. Incubate at 30C until colonies appear (2-7 days). The most spectacular agar degraders will grow in 'sinkholes' in the plates, but given that gar is the only carbon source supplied, if it's growing on the plate, it's eating some kind of agar. Make careful note of colony morphology.
  5. Select a well-isolated colony & streak onto a fresh plate. Repeat the incubation as before.

Observations

agar degrader coloniesmicrocolony

Here is a rod-shaped organism isolated by it's ability to degrade starch (which we've used in the past). The plate shows that it can also degrade agar (note the sunken yellow colonies).