Dear Annie,

There is no requirement to produce inoculate for biofilm experiments by growing up an over-night culture using a rich medium such as LB. You could in fact directly resuspend colony material in 1ml of sterile water (or buffer, or medium) and take aliquots of this to inoculate your biofilm samples.

Sometimes it is important to be able to provide inocula at a certain cell density or in a specific growth phase, and it is very easy to do this in a general growth medium such as LB (assuming that your strain grows well in this). Sometimes it is also important to 'pre-condition' the cells by growing in a medium which initiates a particular biochemical or behavioural response which is required for the assay that will be undertaken, and sometimes it is necessary to make sure that the cells that are prepared are not starved for any particular nutrient ...

We often initiate our biofilm assays with over-night cultures grown in the nutrient-rich King's B media (similar to LB but better for Pseudomonads). An 18h incubation with shaking (inoculated with a wire lop from a plate with fresh colonies) will always result in a good cell density, so we don't have to bother with diluting or concentrating the cells before aliquoting and inoculating the assays. An 18 h culture is also at the start of stationary phase, so cells in the assay cultures won't take long to start replicating fast again. I don't like using 48 hr cultures or older ones (e.g. inoculated late on Friday for a Monday morning start), as in our system a variety of biofilm-relevant mutations can accumulate and spoil our experiments.

Sometimes, however, we want to assay growth or biofilm-formation in a more restrictive media in which carry-over from the over-night culture could be a problem. If we want to assess growth at lower nutrient levels, you might need to wash cells with water or buffer several times (and even allow them to stay in a no-nutrient buffer for a couple of hours) before use to make sure there is no carry-over of nutrients into the new media.

At the end of the day, there is no set way to prepare inocula for experiments. Your aim should be to produce a useable isogenic culture, free of contaminants and contaminating bacteria, and lacking in mutants which might cause a problem. However, if you wish to repeat a published experiment, you really do need to reproduce all of the steps that were used, as small variations in inocula density, cell state and nutrient carry-over, could be enough to cause different outcomes!

Regards, Andrew.

Dear Annie, in our lab, we grow bacteria on tryptic soya agar overnight, then we prepare suspensions in 0.9%NaCl adjusted to McFarland 0.5 using a densitometer. In this way, when we repeat the experiment, we get the same number of bacteria. The presence of sugars especially sucrose in biofilm medium increase biofilm formation in staphylococci.

Dear Annie, starting from overnighr culture is one of several ways to start your biofilm experiment. Due to microbial species and strain peculiarities, there are a variety of approaches for inoculum preparation and also for biofilm growth, coloring and evaluation. You would best make a preliminary optimization trial to choose the appropriate conditions for your strain and the aim of your study.

Due to differences in strains and study objectives, biofilm protocols are difficult if at all possible to standardize, however when you write a publication you should give adequate detail on your experimental protocol. Regarding this, I would very much recommend a recent publication :

"Minimum information guideline for spectrophotometric and fluorometric methods to assess biofilm formation in microplates" (Allkja et al., https://doi.org/10.1016/j.bioflm.2019.100010).

Success in your research!

Stoyanka