Enrichment and Isolation of Thermophiles


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 procedure.

The media used in this experiment will be incubated at high temperatures. Wear gloves to retreive cultures from the incubator, and allow to cool at your bench before manipulating. Handle carefully!


Introduction

The lab is a new experiment, and so don't really know what to expect in terms of results. The media is designed to grow organisms like Thermus, which is reportedly common in hot water heaters, but we'll see....

Although most will think of environments like Yellowstone hot springs or deep-sea smokers when they think of thermophiles, there are lots of microenvironments that are hot at least periodically. It might also be that cooler environment might contin some numbers of thermophiles from other envirnments "chillin' out" on the chance that it might get hotter.

The goal is to isolate organisms that can grow heterotrophically at high temperatures (55°C). The media is just standard LB, made in mineral water instead of distilled water because many thermophiles have higher than usual requirements for some metal ions.


Materials

  • Samples of a hot environment: water from a hot water heater heater clean-out valve, soild from around a steam pipe, or anything else you can think of.
  • Innoculating loop
  • NAM media in tubes and plates:

    Nutrient agar made with mineral water (NAM) is supplied in pre-mixed form - prepare as directed. The only difference is that instead of distilled water, use Pelligrino mineral water

    Note that PYD, TSA (trypticase soy agar), YT, LB, or any other basic rich agar can be substituted for NA for the purposes of this class. Likewise, and high mineral content mineral water can also be substituted.


Procedure

  1. Use a loop to or pipet to add a pinch (or about 1ml if it's clear liquid) of inoculum into a 10ml tube of NAM media. Incubate 1-7 days at 55°C.
  2. When your tube has grown up, vortex your tube and transfer a loopful of the enrichment culture to a fresh 10ml tube of NAM media. Incubate 1-7 days at 55°C.
  3. When your tube has grown up again, vortex the culture and streak a sample onto an NAM agar plate, parafilm the edges tightly, and incubate 1-7 days at 55°C.
  4. Chose a well-isolated colony to restreak onto a fresh plate. Incubate 1-7 days at 55°C.
  5. Examine & restreak as needed until you seem to have a pure culture.

Observations

Make notes on both the colonial and cellular (in wet mounts) morphology. Don't know what we'll get, but these might be excellent candidates for PCR and phylogenetic analysis.