Techniques for culturing bacteria

Isolating rumen bacteria is still of great importance. Newer molecular techniques have established the diversity in the rumen that has escaped cultivation thus far, highlighting the need for renewed efforts in cultivation. Although much can be learnt from community analysis based on examining the 16S rRNA gene or even from metagenomic reconstruction of genomes, much of what we know about rumen function is based upon what has been learnt from isolated cultures. It is especially important to try and isolate representatives of the bacteria that can be detected by molecular means in order to determine their role within the ruminal ecosystem. Validation of predicted activities and interactions are best done using cultured strains. However, the majority of bacteria fail to grow in laboratory conditions. This is typical of many environments, not just the rumen. The development of new cultivation techniques and a better understanding of the rumen environment will potentially help in enabling the isolation of some of these ‘unculturable’ bacteria.

Originally Hungate designed a method to isolate rumen microbes that employed roll tubes, which are essentially a layer of agar within a sealed atmosphere-controlled vessel (Hungate et al., 1973). The oxidation reduction potential of the rumen is very low

(350 mV), and at this potential the concentration of oxygen is less than 1022 M (Hungate, 1975). To culture anaerobic bacteria from the rumen, oxygen needs to be rigorously eliminated from the growth medium. This is assisted by the use of reducing agents like cysteine and sodium sulfide. Anaerobicity is essential, as oxygen is lethal to many rumen bacteria.

From the roll tube method, other methods have evolved to include the use of anaerobic glove boxes and airtight anaerobic jars where an oxygen-free internal environment can be maintained (Cox et al., 1975). With these, media could be used in

agar plate format, which is more convenient for observing and picking bacterial colonies. Isolated bacteria can be grown in anaerobic liquid media in vessels sealed with stoppers that do not permit gas diffusion. Components are transferred into the vessels

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with sterile syringes that have been flushed with O2 free gas (usually N2 or CO2) to remove most of the O2 from the headspace. This enables manipulations to be made under normal laboratory conditions without the need for specialist equipment.

It is difficult to replicate the rumen environment if it is not known which of the parameters are important for bacterial growth. One idea to combat this is to grow bacteria in a diffusion chamber within the natural environment (Kaeberlein et al., 2002).

The diffusion chamber method employs a semi permeable barrier that the cells cannot pass through, but nutrient and growth factors can. It has been shown to dramatically increase the recovery rates of microbes from marine and freshwater environments (Bollmann et al., 2007), but has not been attempted for rumen microbes and would be

technically difficult.

The dilution to extinction method has been used to rapidly isolate a large number of novel rumen bacteria (Kenters et al., 2011). Here 45% of the cultures obtained were

not associated with a known genera (<93% 16S rRNA gene sequence similarity). Button

et al. (1993) established a theoretical basis for this technique, which involves diluting

the sample to be used as the inoculum to the point where it is likely ≤1 cultivable cell is being introduced into the culture vessel. This was shown to improve the cultivation success of marine bacteria (Quang et al., 1998). A single cell cultivation strategy was

able to isolate a greater diversity of anaerobic oral bacteria than conventional plating or a minitrap technique (Sizova et al., 2012). Goodman et al. (2011) used the dilution to

extinction technique to isolate thousands of anaerobic bacteria from human fecal samples in 384-well trays.

One advantage of the single cell cultivation strategy is that it eliminates competition from fast growing bacterial ‘weeds’ that can dominate mixed cultures (Cray

et al., 2013). Dilution theory is used to calculate the appropriate dilution point in order

to have the highest chance of inoculating with a single viable cell while still obtaining a reasonable number of cultures (Figure 1-5). As the number of culturable cells inoculated increases, so does the number of growth positive tubes in that series. If 100% of the series grows, they are statistically unlikely to be derived from a single cell. Using the calculations from Button et al. (1993) to achieve about a 90% incidence of growth-

positive cultures that are derived from single cells, the inoculum should contain an expected 0.2 cells. The number of tubes that will be growth positive will be 20% of the

39 total, and the number of tubes containing cultures derived from a single viable cell will be about 18% of the total.

Figure 1-5. The effect of inoculum size on the number of cultures derived from single cells. The blue line represents the proportion of growth positive cultures as a function of the mean number of culturable cells in the inoculum. The red line represents the proportion of positive cultures that will be derived from a single viable cell. The green line represents the calculated proportion of cultures that will be inoculated with a single culturable cell. Modified from Kenters et al. (2011).

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Recently, high throughput methods to cultivate bacteria have been described. High throughput cultivation is needed to be able to sample the diversity of bacteria present in the rumen. If most rumen bacterial species are present at less than 1% of the population, many cultures will be needed to ‘by chance’ isolate even moderately abundant species. Bruns et al. (2003) described a method using a microdrop dispenser

system to isolate bacteria in liquid media. A microdrop device creates droplets from the bacterial suspension using a similar process to that of an ink jet printer, depositing the droplets into microtitre plates. This was used on bacteria from freshwater lakes but has never been attempted on anaerobic bacteria. The method described by Zengler et al.

(2005) is based on the encapsulation of a single cell followed by flow cytometry to sort microcolonies (originated from a single encapsulated cell) into growth media. Diluted cells are mixed with agarose in an emulsion matrix resulting in approximately 10% of formed microcapsules occupied by a single encapsulated cell. Encapsulated cells can be grown together in media or reintroduced into their natural environment. As they cannot escape their capsules, they remain separate yet in close contact with other cells. This allows cross talk between community members, hopefully including signal molecules and growth factors that enable previously unculturable bacteria to grow. Capsules containing microcolonies can then be sorted and grown. This technique has the potential to isolate 10,000 bacteria per sample, but has never been used for culturing rumen bacteria due to the difficulties in maintaining an anoxic environment.

Soil bacteria have been considered to be difficult to culture. However, it has been shown that three factors are significant for isolating many of the so-called unculturable soil bacteria. These are (1) wide separation of cells in the inoculum to avoid negative interactions, which seem to be more important than positive ones, (2) increased incubation time to allow slower growing species to develop to the point when they can be detected, and (3) screening of large numbers of cultures or colonies to allow detection of species that may be abundant in the starting material but may initiate growth under laboratory conditions only rarely (Davis et al., 2005; Davis et al., 2011;

Janssen, 2008; Joseph et al., 2003). These approaches could be applied to the rumen to

increase the likelihood of isolating part of the missing diversity.

It is desirable to combine molecular biology with microbiology and examine genomes as well as cultured organisms to get a more complete picture of the processes

41 occurring within the rumen. A modern approach to cultivation is to use knowledge gained from culture independent studies to target the culturing of groups of previously uncultured bacteria. Koike et al. (2010) used quantitative real time PCR and FISH to

monitor the enrichment of an uncultured group of bacteria designated U2, and specific PCR primers to screen isolates for members of the U2 group. In this way, two isolates were identified (R-25 and B76) of a group that was previously only represented by 16S rRNA gene sequences obtained from culture-independent studies. These organisms are now available for further characterisation and may prove to be an important fibre degrading group in the rumen.

Molecular techniques can also provide clues on how to culture unknown bacteria. For example Bomar et al. (2011) used high-throughput sequencing of RNA transcripts

(RNA-seq) to characterize the metatranscriptome from the leech gut environment. They determined the dominant uncharacterised organism was utilizing mucin, thus allowing its subsequent cultivation on a medium containing mucin. Pope et al. (2011) directed

the isolation of a novel succinate-producing bacterium from the wallaby gut using reconstructed bacterial metabolism from metagenomic data. These principles could also be applied to bacteria from the rumen.

New culturing techniques involving microscale cultivation using hollow fibre capillary membranes or microfluidic chips for separating many bacteria in a small volume are being developed (Stewart, 2012) and are promising approaches for the future if they can be adapted to anaerobic environments.