Several Escherichia coli (ATCC 11229) bacterial cultures were established using serial dilutions and simple agar plate pouring. The growth of the bacterial cultures was evaluated using spectrophotometric and colony counting methods. Data collected from a two-hour monitoring using 30-minute interval sampling of bacterial suspensions were plotted against the 30-minute interval collection times on a normal and semilographic coordinates. The resulting growth curves showed that the cultures progressed from the lag to the log phase, which are typical of a bacterial growth curve.
The growth curves generated by spectrophotometric analysis were similar to the growth curve created by the colony counting method. The techniques employed in this experiment may serve as basis in determining bacterial growth on both liquid and solid culture media using other types of bacterial species. The methods used in this exercise may act as a method in estimating the number of cells that are generated through different types of culture conditions.
The colony counting method seems to be a more reliable method because it involves actual visual inspection of live colonies while the optical density readings involve light transmission through the turbidity of the bacterial culture suspension. The growth of bacteria pertains to a process wherein a single bacterial cell generates two identical daughter cells. This simple doubling of bacteria is observed in cultures that are classically conducted in microbiological laboratories. The quantification of bacterial growth is generally performed through the use of either direct or indirect cell counting methods.
Colony counting is an example of a direct counting technique while the measurement of turbidity is an illustration of an indirect counting procedure. The progress of a bacterial curve is generally described through the use of a growth curve (Novick, 1955). Four different phases comprise a bacterial growth curve. The lag phase involves the adaptation of inoculated bacteria to the conditions of the culture medium. This phase denotes that time that the bacteria are undergoing maturation. The logarithmic or exponential phase involves the doubling of bacteria in culture.
The rate of division is observed to logarithmically increase through time. The growth conditions and the chances of survival of the resulting daughter cells influence bacterial growth rate. The logarithmic growth of the bacterial culture is dependent on the availability of nutrients in the culture medium. The stationary phase pertains to the decrease in growth rate due to the exhaustion of nutrients in the culture medium and in turn, wastes have accumulated in the culture medium. During the death phase, the cultured bacteria lose nutrient resources and die. Materials and methods. The bacterial concentration of an E.
coli culture (ATCC 11229) broth culture was determined through the employment of serial dilutions and agar plate counts. Approximately 1 ml of the E. coli culture at log phase was transferred to a flask containing 100 ml of brain heart infusion broth. The suspension was slowly swirled and 5 ml was transferred to a cuvette for optimal density (OD) reading at 600 nm absorbance. Another 1 ml of the log phase bacterial culture was transferred to a test tube containing 9 ml of water. The suspension was mixed well then 1 ml of was then transferred to another test tube containing 9 ml of water.
The serial dilution was performed six times, resulting in 7 dilutions. Approximately 1 ml of the 0 time point dilutions (10-4, 10-5, 10-6, 10-7) was plated with 15 ml of melted agar, swirled evenly and set aside to solidify. The optical density (OD) of the broth culture was taken every 30 minutes by transferring 5 ml of the broth culture to a cuvette for spectrophotometric reading. All plated cultures were incubated for 24 hrs at 35-37oC. After 24 hrs incubation, the colonies that emerged on each plate were counted. The collected data from the OD reading and colony counting were then analyzed and plotted on semilog paper.
Calculation for generation time (g) of the bacterial culture was performed using the following equation: g = time at absorbance 0. 41 – time at absorbance 0. 21 Results. The growth of the bacterial culture based on the concentration of bacterial cells was determined using two methods. Optical density (OD) reading using the spectrophotometer showed that the number of bacterial cells in the culture increased for the 2 hours that the cultures were monitored. The optical density reading were then plotted against the 30-minute interval collection times (Figure 1).
Based on the collected data, the calculated generation time is 32 minutes. The generation time was determined as follows: g = 92 minutes – 60 minutes = 32 minutes Data collected from colony counting of broth cultures were plotted against the 30-minute interval collection times on a semilographic coordinates (Figure 2). The generation growth curve was determined to be 10. 1 minutes, indicating that the bacterial culture had grown 10 times from the start of the experiment. The exercise involving determination of bacterial concentrations over 2 hours of monitoring showed that the broth culture of E.
coli showed an increase in its growth. The data showed the features of the lag and logarithmic phases of a growth curve wherein there is a slow adaptation stage that shows minimal increase in the number of bacterial cells during the first 30 minutes of inoculation and then an exponential increase in the number of bacterial cells was observed soon after until the end of the experimental period. The results generated from optical density readings and colony counting show the same increasing trend in the number of bacterial cells in culture.
The employment of two methods in determining growth rates of bacterial cultures provided an opportunity for comparison of these methods. The colony counting method seems to be a more reliable method because it involves actual visual inspection of live colonies on a Petri plate while the optical density readings merely involve light transmission through the turbidity of the bacterial culture suspension and this method does not differentiate the live from the dead bacterial cells but in turn just counts the number of bacterial cells that are present in the cuvette.
It should be noted that the number of viable bacterial cells serve as the source of new daughter cells hence it is better to rely on the results that are generated by actual colony counts derived from visual inspection of Petri plates.
However, it also should be noted that colony counting is also associated with a disadvantage wherein one colony may be composed of at least two to several bacterial cells hence the exact number of cells can not be determined. A colony composed of more cells can thus result in a bigger number of daughter cells than a colony comprised of only 2 bacterial cells. Reference Novick A (1955): Growth of bacteria. Annual Review of Microbiology 9:97-110.
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