Doubling the divide and tackling the S-curve: How bacteria grow

Bacteria, and some yeasts, grow and divide by a process known as binary fission (they will grow continually until, on reaching a critical size, they divide into two equal-sized parts - the splitting of a unicellular organism into two). Each cell increases in size until it has doubled its mass. A dividing cross wall is then produced which splits the cell into two identical daughters. Each of these daughter cells will go on to grow and divide further (1).

In this week’s newsletter, I’m looking at one of the simpler, back-to-basics areas of microbiology. The content will be familiar to experienced readers, but hopefully of interest to those new to the field and my non-microbiologist readers.

Given appropriate supplies of nutrients and ideal growth conditions (pH, temperature, water availability etc.) then the division process can occur very frequently (for example, 3-4 times per hour). This leads to a rapid and exponential increase in population size. An impression of this growth potential can be gained with the following example.

Example of bacterial division

An organism of 1 µm diameter divides every 20 minutes (this is the bacterium’s generation time). Starting with a single cell there will be eight cells after 1 hour, 64 after 2 hours… and then 10^8 (or 100, 000, 000) at 10 hours and 10^12 (a million million) at 15 hours. With 10^12 bacteria, of this size, the bacteria will have a combined volume of 1cm^3. However, in the real world such masses are rare due to the availability and type of nutrients in natural ecosystems, which tend to prolong the generation times from anything from 20 minutes to l-10 hours.  Hence, there are natural limitations to the total biomass (2).

To put biomass in context, a population of 1 million cocci will fit side by side on the surface of a pin-head (1mm^2). If these cells stack to form a pyramid-like structure, then the numbers could exceed half a billion. If we consider that non-sterile pharmaceutical and cosmetic products are typically required contain less than 10 organisms per gram of solid and less than 100 organisms per ml of liquid then this illustration signals how small, overlooked, foci of bacterial contamination can lead to contaminated products (3).

Growth and division of bacteria in a liquid medium

A typical growth curve, for a bacterial population inoculated into a closed container filled with a liquid nutrient medium (as would commonly be undertaken in a microbiology laboratory). Growth will become evident during the latter stages of this process as an increase in the turbidity of the liquid. The maximum specific growth rate (µ max ) and the lag phase (λ) are key parameters (4).

The chart below shows the classic sigmoidal (‘S’) curve for bacterial growth:

Lag Phase

The bacteria do not start to grow immediately. The cells undergo an adaptive phase whilst their metabolism is altered to suit the availability of nutrients in the flask and also to suit the pH, osmolarity and temperature. This is the lag phase. It can be as short as a few minutes, if the cells have recently been growing in a similar medium, or it can be extended for up to many days or weeks. Extended lag phases will result from the transfer of small numbers of cells from a nutritionally rich environment to one that is nutritionally poor. In pharmaceutical and cosmetic products the lag periods may extend long after manufacture such that bacterial growth becomes apparent during storage.

Exponential (logarithmic) Phase

Once the cells have become adjusted to the physicochemical conditions in the liquid then the population increases in an exponential fashion (above). The population growth rate (µ) is maximal at this time and is related to the generation time (GT) of individual cells:

[µ = ln2/GT]

The exponential phase of growth may only be short lived since as the cells grow they will consume nutrients and produce waste products. The latter will often include organic acids which will lower the pH of a bottled product. Consumption of nutrients or changes in the pH of the product will reduce the rate of growth (late exponential phase) and eventually stop it completely (stationary phase).

Stationary and Decline Phases

The density of cells in the liquid at the onset of the stationary phase will depend on the amounts of utilizable carbon and nitrogen source present at the outset. In nutritionally poor vehicles, such as distilled water, where nutrients are scavenged from the container walls and from the air, then the population density at this point might be very low and in the order of 104 cells/ml. In nutritionally rich environments (which would include things like oxtail soup and dextran solutions) then the numbers are likely to be much higher (around 10^9-^10/ml). It is worth bearing in mind that bacterial counts of 10^6/ ml in water are barely visible to the naked eye (if possible at all) and might form within a few hours of the water being placed into a soiled or non-sterile vessel.

If the stationary phase is induced through a lack of carbon source (carbohydrate, sugar etc.) then the cells will be unable to survive for an extended time period. The majority of the cells present will die and release releasing nutrients which will enable the remainder to survive. This is called the decline phase. The extent of the decline phase will depend on the size of the population achieved at stationary. If this is high then the remaining carbon reserves will be insufficient to sustain the population for long. If the numbers are low then the decline phase will be delayed and extended in its effect. Low numbers of survivors may remain viable for many years.

If the stationary phase is brought about through a shortage of available nitrogen (protein, amino-acids etc.) and carbon substrate remains in excess then the entire population may remain viable for an extended time period until the carbon reserves are also consumed.

Endospore forming organisms such as the Bacilli and Clostridia commence sporulation at the onset of stationary phase. Sporulation may be completed within 5-10 hours. The spores produced will remain dormant and viable, irrespective of the further availability of nutrients, for many years.

Growth on solid surfaces

When bacteria or yeasts come into contact with a solid surface which is both nutritious and moist then they will undergo a period of growth as described above for liquids. On solid surfaces, however, the nutrients are not readily available to all cells and these will be unable to diffuse away from one another.

On an agar plate, the result is that the cells produced as a result of division build up on the surface to eventually produce a colony which is visible to the naked eye (1 - 7mm diameter). A gradient of nutrients is created within the colony as nutrients diffuse into it from the surface and are consumed preferentially by those cells adjacent to it. The diffusion of nutrients ultimately restricts the doubling time of the colony. In this manner the growth of colonies is diffusion limited and, in contrast to liquid cultures, will be maintained for many days.

The ability of microorganisms to form visible colonies on solid nutrients is exploited by microbiologists in order to estimate numbers of viable bacterial cells. Nutrient-rich broths are solidified with agar. This provides a solid gel surface, rich in nutrients and with high water activity. If a suspension of bacteria is spread onto the surface of an agar plate (contained in a Petri dish), then each viable cell will be capable of forming a single colony.

Counting the colonies (colony forming units, CFU) then gives an approximation to the numbers of viable cells in the original suspension. Alternatively the agar plates may be exposed to the air in which case the colonies reflect the organisms railing onto the surface of the plate during exposure (settle plate). Swabs taken from surfaces may similarly be spread onto agar plates to give an indication of the extent and nature of the contamination.

Recognition of microorganisms is also made possible through the use of such media. The size, shape, color, texture, smell, reflectivity and cross section of colonies differs between growth media and species. This, together with a knowledge of the incubation conditions and the type of medium, enables microbiologists to recognize different species and to separate and isolate single organisms from mixtures of bacteria.

Organisms isolated as single colonies can, in addition, be subjected to microscopic examination and/or biochemical tests in order to establish their identity. Solidified nutrient media, containing specific chemical growth inhibitors have been designed which allow the growth only of certain specified organisms. These are called selective media and are used to screen and monitor for particular problem organisms. The use of different media for the isolation and detection of microorganisms in pharmaceutical products provides the basis for the Microbial Limits Test.

Tim Sandle’s  new book  “Biocontamination Control for Pharmaceuticals and Healthcare” 2nd Edition is published during March 2024 and it is available for pre-order.

References

1.      Adams DW and Errington J (2009) Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nature Reviews Microbiology 7: 642–653

2.      Leaver M, Dominguez-Cuevas P, Coxhead JM et al. (2009) Life without a wall or division machine in Bacillus subtilis . Nature 457: 849–853

3.       Margolin W (2009) Bacterial division: a new way to box in the ring. Current Biology 19: 881–884

4.      Zwietering, M.H., Jongenburger, I., Rombouts, F.M., Van’t Riet, K., Modeling of the Bacterial Growth Curve. Appl. Environ. Microbiol. 56, 1875-1881 (1990)