Anaerobic Growth

The cultivation of strict anaerobic bacteria poses a special problem because these bacteria may be killed by exposure to air. Dissolved oxygen in the medium forms toxic free radicals and hydrogen peroxide in the presence of metabolic electrons. Obligate anaerobes are incapable of detoxifying these active forms of oxygen. To grow non stringent anaerobes on solid media, anaerobic jars are used together with gas generating "Gas-Paks", which release both CO2 and H2. The hydrogen reacts with oxygen in the presence of a palladium catalyst to produce water, thus removing oxygen from the jar. Completely anaerobic chambers equipped with air locks and filled with inert gases used for the cultivation of strict (obligate) anaerobes are commercially available.

Redox Potential (O/R Potential)

This is the proportion of oxidized to reduced molecules in a medium: when oxygen dissolves in a medium, for instance, the organic compounds present become more oxidized and the medium exhibits a positive redox potential. As microbial growth consumes the oxygen, the medium moves towards a more negative redox potential. Strict anaerobes require the medium to be kept at a very low (negative) potential during growth. To achieve this, reducing agents are added to the media prior to autoclaving. Commonly used reducing agents are sodium thioglycolate (HS-CH2COONa) or sodium dithionite, which easily donate protons to other compounds. The relationship between redox potential, pH and microbial growth is illustrated below.

Eh and pH ranges for microbial growth (adapted from Zajic 1969): the figure has been compiled from reports where both pH and Eh were given for growth behaviour. It is very probable that the growth ranges of the groups extend beyond the boundaries shown in the figure.

Monitoring Microbial Growth

Serial Dilutions

The inoculum is diluted out in a series of dilution tubes which are plated out. The number of colonies on the plate are counted and corrected for the dilution to calculate the number of organisms in the original inoculum.

Most Probable Number Method

A statistical method estimates the most probable number of bacteria present in an inoculum which has been used to make a dilution series. Several series are made with different initial volumes of inoculum; the results are recorded as a series of positives, i.e. growth in the tube, which can then be calculated to give an MPN. The result is the probable number of microorganisms that would be expected to yield this result.

Direct Microscope Observation

Specially constructed microscope slides are used which have a shallow well of known volume and a grid etched into the glass. The well is filled with the bacterial suspension and the average number of bacteria in each of the grid squares is determined and then multiplied by a factor to give the counts per millilitre. Selective staining (employing fluorescent dyes) is used to differentiate bacteria from non-living material in environmental samples. Electronic cell counters are also available which automatically count the number of cells in a measured volume of liquid.

Turbidity (Optical Density)

The turbidity of a liquid medium increases as bacteria multiply and can be measured on a spectrophotometer. The amount of light reaching the detector is inversely proportional to the number of bacteria under standardized conditions. The absorbency of the sample (optical density) is dependent on the number of cells, their size and shape, and is used to plot bacterial growth. If absorbency readings are matched with a direct count of the same culture, its protein content or dry mass, the correlation can be used in a future estimate of bacterial numbers or biomass based directly on turbidity measurements.

Metabolic Activity

Another indirect way to estimate bacterial numbers is to use the metabolic activity of the population. The amount of a metabolic product is measured and assumed to be proportional to the number of bacteria present. Examples of metabolic products include CO₂ and organic acids. Oxygen uptake can also be measured with a reduction test, for example the use of the methylene blue dye, which changes colour from blue in the presence of oxygen to colourless in its absence.

Preserving Bacterial Cultures

Refrigeration

Can be used for short-term storage. Cultures streaked on agar slants or stab cultures may be viable over several months when stored at 4°C. Plates have to be sealed to prevent their tendency to dry out. To preserve cultures for longer periods of time, two methods are commonly employed:

Deep Freezing

A pure culture of bacteria is suspended in a liquid and quick-frozen (often with liquid nitrogen) at temperatures between -50°C and-95°C. Sensitive microorganisms require the presence of glycerol (end concentration 15-20 %), which acts as an "antifreeze", or extra protein (skimmed milk powder) to protect them. Cultures can be thawed and used up to several years later.

Lyophilization

A suspension of bacteria is quickly frozen and the water removed by means of a high vacuum. The microbes survive in thes powderlike residue for several years and can be revived at any time by rehydration of the culture in a nutrient medium. Bacterial strains ordered from strain collections are usually delivered in this form.