Moore:Working with coli

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Revision as of 15:40, 2 July 2008


The Bug

E. coli is a Gram-negative bacterium classified as γ-proteobacteria. The closest to "wild-type" strains are probably a clinical isolates, but in the lab "wild-type" usually refers to a parental strain to which mutants are compared. Under ideal conditions (like in a fermenter), E. coli can replicate about every 20 minutes, but you are more likely to see a healthy strain divide about every 30-35 minutes in a well-aerated liquid culture at 37 °C. Most lab strains are considered harmless to human health (BL-1).

E. coli will grow at some rate anywhere from from ~15-45 °C until it has used all of an essential resource. After that, the bacteria enters a stationary phase wherein metabolism is greatly reduced and a series of physiological changes occur as the organism attempts to stay alive. Given enough time (a few days at room temp), a significant fraction of the bugs will die of starvation.

Growing It

For most applications, the bacteria are growm in a medium containing yeast extract and tryptone as food sources (like LB broth). While this is an affordable and convenient way to make a lot of cells, care should be taken if these media are used during experiments. The pH and available carbon, nitrogen, and mineral resources are not controlled in these preparations so you can observe significant differences in behavior of the cultures from experiment to experiment. For controlled experiments, it is better to use a rich, defined medium that has all of the necessary components for growth. A buddy of mine and I set up an experiment to see how dense a culture would get if resources were not limiting. We put a healthy lab strain in a dialysis bag and dialyzed the culture agains fresh growth medium every day. After a week, the culture had stopped getting denser: when we measures the density of cells, the "OD" (described later) was ~85. The culture was still liquid though (a thick one) so the cells were not as dense as they are in a colony on a plate.

A Note On Antibiotic Selection

Many times, you will have a need to maintain genetic elements that are linked to a gene that confers antibiotic resistance (for example a plasmid harboring a gene you want to express containing a "drug marker"). You should always keep in mind what the selection antibiotic is, how it inhibits bacterial growth and what the resistance gene does to allow growth.

Storing It

Most E. coli strains are stored as "glycerol stocks" at -80 °C. To make these, liquid cultures are mixed with glycerol (10-20% final) and frozen. A scraping of the "freezer stock" is then used to seed fresh cultures as needed. In the absence of a cryo-protectant (like glycerol), freezing will rupture the cell membranes and kill the bug. In fact, freeze/thaw is one method to promote cell lysis for extracting protein. After new strains are generated, it is a good idea to make feezer stocks from relatively fresh cultures. If you need to wait for a few days (perhaps for sequencing data), keep the cultures at 4 °C.

Killing It

E. coli is a pretty easy bug to kill. Most laboratories have defined protocols for sterilizing cultures/colonies before they are disposed of, so be sure to make sure your safety officer is happy with whatever you choose to do. Some of my favorite methods: bleach, autoclave, 75% ethanol, Wescodyne, and flame.

Measuring Culture Density

The simplest way to monitor a culture density is to measure the turbidity or "absorbance" (A) of a culture at a wavelength of light in the visible range. The reason I put absorbance in quotes is that it is a classic misnomer: the wavelngths of light chosen to measure cell density are deliberately chosen in ranges where absorbance is negligible. What you measure is the attenuance (D) of light as it passes through the sample before it reaches the detector. The attenuance is a combination of all of the light lost from absorbance, scattering, and instrument geometry. You won't be able to win over an editor when it comes to labeling your growth plots with D, just be aware that the "A600" measured in one spectrophotometer can be significantly different for the same cell density in another instrument. It is a good idea to measure the growth of a culture in the spectrophotometer you will be using so you are aware how the readings relate to the growth phase of the bacteria. You may go so far as to plate dilutions of the cells at different "absorbance" values to be able to convert your data to colony-forming units (c.f.u.).

Another common way of reporting density is with the term "optical density" (OD). Generally, this refers to the apparent absorbance of the sample in a 1 cm path cell. There is a really confusing tendancy of people to report amounts of material (cells, DNA, protein, etc.) as "ODs". When you see this, what is meant is "1 ml of solution with an absorbance of 1 per cm". Why they don't use a concentration term is beyond me.

There is no reason to stick to using only 600 nm as the wavelength for measure culture density, any wavelength the scatters in proportion to the concentration of cells is fine. You may want to use 450 nm when the culture is very dilute, and 700 nm when it's thick. You can re-scale values measured at one wavelength to another value quite easily. Rather than fitting your data to a Mie scattering formula, you can make an empirical curve for one culture and use the relative values at different wavelengths to adjust values at other wavelengths.

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