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Following growth of E. coli cultures

24 October 2019 Tags: spectroscopy growth curves Escherichia coli E. coli OD600

The conventional cuvette-based way of measuring the OD600 of E. coli cultures comes with the major problem that the culture volume is diminished every time a sample is taken. In addition it uses cuvettes, even though they are cheap, and it is hassle. In the lab of Bob Lloyd we used the much better technique of measuring the OD in the tubes we were growing the cultures in. By using a Spectronic photometer with a round sample holder, our 1.8 cm diameter culture tubes were fitting perfectly. Obviously this technique is not particularly accurate, as the light path exceeds the conventional 1 cm path length and any scratches on the glass tubes will cause additional scattering. However, for most applications this "non-invasive" technique is perfectly adequate, as it is very quick, the samples remain perfectly sterile and no culture is lost. For procedures that require a high accuracy cultures can be grown to approximately to correct OD and additional measurements can be taken with a cuvette if necessary.

Jenway 7300 spectrophotometer with specialised insert

Unfortunately when I set up my own lab the old Spectronic photometers were not available any more, especially with the convenient round sample holder. While I was on the hunt for a used machine I was contacted by Tessa Hargrove, who at the time was a sales representative for Bibby Scientific. When we talked about my particular problem she went through great lengths to see whether a different spectrophotometer could do the job. It came down to quite some tests and only about 5 mm, but in the end the at the time new Jenway 7300 spectrophotometer could be modified to do the job. The Jenway 73XX series comes in a variety of "flavours" to allow measurement with UV and visible wave lengths. It is a relatively bulky machine with quite a large footprint – for our particular purpose this fits very well, as it needs to hold an entire culture tube.

The standard model comes with a conventional holder for 1 cm cuvettes – obviously far too small to fit our 1.8 cm diameter culture tubes. However, after some enquiries Tessa found a larger sample holder. The hole is slightly too large, but with a small rubber wedge tubes can be held safely. Measurements are, in fact, slightly more consistent than with the Spectronic, because the measuring chamber is closed and no stray light can cause problems. I spent several days recording growth curves and plotting the relation to measurements with a 1 cm path length and I was surprised how accurate measurements are if performed correctly. Obviously scratches on the glass remain a problem, but we routinely sort out badly scratched tubes and, as before, for cruicial experiments a separate reading can be taken with a 1 cm cuvette once the culture has reached approximately the right density.

This means that we can still use the fast and efficient way of growing cultures as was done in Bob Lloyd’s lab. We use standard rimless test tubes provided by SLS with OXOID aluminium caps – we were just about able to buy 200 of these before stocks ran out. I find it a shame that OXOID is not producing these any more, as the plastic alternatives from Fisher are nowhere near as good as these. Cultures are grown in Grant OLS200 shaking waterbaths with a specialised insert that Grant Instruments made to our specificiation with clips provided by Springmasters. Culture tubes are held at an angle, which increases the culture surface and allows for excellent airation – our E. coli MG1655 reference strains grows with doubling times just below 20 min. I find the entire setup efficient and very convenient.