Thursday, 3 January 2012

09:45 – I’ve been dithering about whether or not to include a mixed bacteria culture in the Life Science kits. Well, not literally a culture, because a culture is by definition reproducing. Such cultures are provided in a nutrient broth or on a nutrient agar slant, and require special handling, often including refrigeration. They’re normally shipped next-day air and must be opened and used within a couple days after they arrive. Even allowing the culture to sit undisturbed over a weekend can cause problems. The problem is that cultures continue reproducing until they run out of nutrients and become senescent. Mutations occur, and eventually the culture becomes useless.

Other than freeze-drying a culture, there are two ways to avoid that. First, one can reculture every few days to every few weeks, transferring a small amount of the culture to fresh media, and then repeating the reculturing as necessary to maintain a robust culture. That’s obviously not practical for pre-packaged science kits. Option two is to put the bacteria into stasis (essentially, hibernation) by inoculating either a sterile saline solution or a sterile PBS (phosphate-buffered saline) solution with the bacteria. The saline/PBS contains no nutrients, so the bacteria don’t reproduce. Stored in the dark at room temperature, such saline/PBS specimens may remain viable for anything from a few years to many decades, depending on the particular bacterial species and other factors.

The problem is that I don’t have years to decades to find out which species are suited to stasis, and there’s not a whole lot in the literature other than for pathogens. I’d like to provide a mixed group of non-pathogenic bacteria that encompass the three basic morphologies as well as examples of Gram-positive and Gram-negative species. It’d also be nice to have an example of a species that is a facultative anaerobe. On that basis, I’ve tentatively chosen Bacillus subtilis, Micrococcus luteus, and Rhodospirillum rubrum.

So, here’s what I think I’m going to do. Make up and autoclave a liter of saline or (probably) PBS. Using aseptic procedures, transfer about 5 mL of a robust mixed culture of those three species in nutrient broth to the 1 L of sterile saline, mix, and then fill 200 sterile 15 mL polypropylene centrifuge tubes to 5 mL each. Recap and tape each of the tubes, label them, and store them in the dark at room temperature.

Worst case, at least a few individuals of each species should survive statis, so reculturing in nutrient broth or on a nutrient agar slant or plate should produce colonies of each of the three species. Of course, I may be expecting too much of 7th or 8th grade students, not many of whom are very skilled in aseptic procedures. I suspect many, even most, of the tubes will end up contaminated with environmental bacteria, but I’ll have done what I can do.