Pathogenic leptospires are often cultured in the laboratory, both for research and as part of tests for determining infection in a patient – however they are some of the most difficult bacteria to culture and some of the standard techniques may not always work. The most important factor is to replicate the same conditions found in-vivo, as many pathogenic strains will only show positive growth curves under those conditions.
Subculturing usually transfers a few ml of innoculant, and must be done with full biological isolation (no mouth pipettes!). Equipment can be autoclaved or washed in acid and dried. While solid or slush cultures can be stored in the general environment, liquid cultures with agitation should be stored in an isolation cabinet, and procedures conducted with isolation in place. Cultures of leptospira all look the same, so labelling is critical if there are several batches in the lab at once! Also, since the bacteria are such difficult colonies to grow, contamination is a nightmare and extremely careful preparation of containers and media is essential.
Temperature
Pathogens grow best at around 29-32°C but can be cultured up to 39°C. Colonies that have been extracted from a host tend to do best at the host’s body temperature, but colonies kept ex-vivo will revert to the lower temperature range. Pathogens will not grow below 13°C but saphrophytes will, so reduced-temperature cultures can show the pathogenic/non-pathogenic nature of a sample.
pH
All species prefer the narrow pH window of 7.2 to 7.6, and usually fail to proliferate in acid conditions.
Aeration
CO2 is essential to trigger growth and the bacteria are aerobic, so oxygen is required and cultures benefit from gentle aeration or shaking – but some of the really annoying strains react badly to excessive aeration, so a gentle orbital agitator is often the best plan, rather than spurging.
Doubling time
Pathogenic leptospires have a very long fission time, and under the best conditions in-vivo and ex-vivo we expect a 6 to 8 hour doubling interval. More typically we see an interval around 16 hours. There is also an extended lag time, with cultures in-vitro often taking several days before any growth is noticeable.
Lysis
A culture may take several days to grow, and density may increase more slowly than for other bacteria, but the primary concern for cultivating samples is concentration-induced lysis where the lipases produced by the bacteria begin to outweigh the absorptive ability of the culture, and toxic lipids form. This can be a very rapid effect either during the last portion of the log curve or the stationary phase, and can of course play havoc with cultures if they are not subcultured and diluted often enough.
Liquid culture media
Liquid media is the standard for leptospiral culture, and the bacteria seem to prefer it. Maximum concentrations are smaller than in other bacteria, and often reach only 107/ml or 108/ml – when the medium will have an obvious turbidity. Leptospires are extremely small and highly motile, so cultures that are not stirred will not settle out – though there can be precipitation of waste and nutrient material. For reasons we do not entirely understand, leptospires can alter the structure of haemoglobin and so in cultures containing visible coloration from haemoglobin the leptospiral growth can create a color change, moving from pink to brown. Other colors are a sign of contamination. It can be a benefit to use quite a high innoculum, as several pathogenic strains fail to develop a positive growth curve unless the initial colony is substantial – innocula of up to 10% volume can be needed for particularly annoying strains, especially where the innoculum contains only a small proportion of invidivuals adapted to ex-vivo conditions.
Slush culture media
Semi-solid media, such as 1:1000 agar, will develop colonies under the surface and can be used for preservation and physical immobilisation of samples for research – it appears that the bacteria cluster around particles of agar in the slush, using it as a nutrient source, and so do poorly on the surface even if innoculated there. Redox indicators can be used to predict growth positions a day or two in advance of the colony being visible.
Solid culture media
In more solid media such as 1:100 agar, colonies will also grow under the surface but extremely slowly. The medium must be protected against dehydration, and even with the best temperature and gas conditions it can take several weeks to see growth with the naked eye. Growth starts from a point and develops into a dense colony of varying size and shape, and again a redox indicator will blanch a day or two before the colony is detected visually – the first signs are usually tiny 1mm or 2mm diameter dots just under the surface. It’s worth paying attention to the edges of the dish and to any included bubbles, as the bacteria seem to appear there first – indeed having a ‘bubbly’ gel by intention can be a good idea.