Bacterial Growth Patterns Practical Report

BACTERIAL GROWTH PATTERNS PRACTICAL REPORT

Introduction

The growth of bacterial populations relies heavily upon the availability of optimal environmental conditions, such as a suitable temperature range, availability of nutrients and oxygen, and the pH of the surrounding medium. Factors such as the accumulation of metabolic waste products also have an impact on the population density. Similar growth patterns are observed in other microbial organisms such as Fungi, Protozoa and Algae.

Temperature of the surrounding environment is important for optimal growth in bacterial populations. Whilst most bacteria have an optimal temperature ranging from between 25-40oC (mesophiles), there are others that thrive in extremely high temperatures greater than 45oC (thermophiles) or in extremely cold temperatures less than 15oC (psychrophiles). Extremophiles is the name given to bacteria that have an optimal range of between 100-120oC, usually found living at the bottom of the ocean in thermal vents.

Bacterial growth is affected by the pH of its environment, as each population has a certain range for optimal growth. Most natural environments have a pH value ranging from 5-9, with many bacterial organisms having an optimum in this range. However, acidophiles, acid-tolerant bacteria, grow well in particularly acidic environments (less than pH 4), and bacteria that flourish in alkaline environments (greater than pH 10) are known as alkaliphiles. When bacteria are grown in batch culture (a closed system where only a limited supply of nutrients required for bacterial growth is provided), only a few generations grow until final nutrient depletion. As this occurs, toxic waste products accumulate, changing the pH of its environment from its optimum, further decreasing cell growth.

Oxygen is another key factor involved with bacterial growth. It is either a fundamental requirement or a lethal product. Bacteria are divided into particular classifications based on their oxygen requirements: obligate aerobes (oxygen is required for survival), obligate anaerobes (growth does not occur in the presence of oxygen), facultative anaerobes (growth occurs in the presence as well as absence of oxygen), facultative aerobes (growth occurs in the presence and/or absence of oxygen, but grow more rapidly in aerobic conditions) and microaerophillic (grow best in low oxygen concentrations).

Bacterial populations in batch cultures have certain growth patterns that can be divided into four distinct phases. The first phase observed is the lag phase; in this phase no growth is detected. However, bacterial cells are metabolically active in their quest to becoming accustomed to a new environment. The lag phase itself is where bacteria synthesize essential proteins and DNA required for the next phase, which is known as the log phase. This phase is where the rapid growth of the bacterial colony occurs, and the number of cells increases exponentially. Cells here are doubling at a continuous rate known as the "generation time"; the population doubles each generation. The log phase is followed by the stationary phase. Here, the growth of the population begins to slow down and reach equilibrium, as the rate of cell growth becomes equal to that of cell death. Bacteria do not grow to a high population density due to the fast exhaustion of its limited resources as well as the build up of toxic waste products. The final stage of growth is represented by the death phase, where the rate of cell death exceeds the rate of cell growth. A decline in the population is observed here as cells quickly lose their ability to divide. This kind of exponential growth of bacteria can be plotted on a logarithmic scaled graph, with time versus the log of cell number. From the graph, the growth rate constant (the rate of cell division in a culture) can be determined.

The four growth phases are easily recognizable in a logarithmic graph.

Fig. 1 The four growth phases of a bacterial population.

There are many laboratory techniques that are mainly used for the quantitative calculation of microbial populations. Three common techniques used, from the most sensitive to least, are viable count, total cell count and turbidometry. The viable count method gives a quantitative knowledge of the population density of living cells. It is supposed that a single progenitor cell has given rise to one colony only, and the expression of 'colony forming units' is used to justify the fact that a colony may have arisen from two or more progenitor cells. The total cell count comprises of using a haemocytometer - a specialized microscope slide that that contains a counting chamber and a known amount of volume. Here the number of cells, both dead and living, is counted in a known volume. The simplest method involves measuring the turbidity of a cell suspension by using a spectrophotometer. The progressive degree of turbidity/cloudiness of a bacterial culture is measured by scattering light through the sample; as there is an increase in the cell population, the amount of light transmitted through the spectrophotometer decreases. The absorbance of each recording is noted.

In this experiment, turbidometry is the quantitative technique that is used to determine the fluctuating growth patterns of Vibrio natriegens in two different conditions; aerated and non-aerated.

Aims

* To use turbidometric technique to observe and identify the distinct phases of bacterial growth

* To see the effect of different growth conditions in bacterial cultures

Materials

As per lab manual (Monash University MIC2011 - Introduction to Microbiology and Microbial Biotechnology, Exercise 9, pg 73)

Method

As per lab manual (Monash University MIC2011 - Introduction to Microbiology and Microbial Biotechnology, Exercise 9, pg 73)

Results

Fig. 2 The growth curve of V. natriegens from two samples from aerated and non-aerated conditions.