Bacterial Growth

Introduction

Bacterial growth is when the number of bacterial cells multiplies that is an increase in the bacterial population size with each cell having identical DNA similar to the parent cell. Bacteria usually go through asexual reproduction which is binary fission. Binary fission will produces two genetically identical daughter cells (Pommerville, 2010).

There are four phases in the bacterial growth curve that are the lag phase, log phase, stationary phase and death phase. The lag phase occurs when the cell does not go through cell division yet as it adapting to the new environment (Pommerville, 2010). The metabolic activity of the bacterial cells will determine the length of the lag phase. The bacterial cells prepare to go through binary fission by growing in size and synthesizing important enzymes. The next phase which is the log phase is the bacterial population enters a growing stage. This phase is when the bacterial cell will go through binary fission and the environmental factors will affect the generation time of the bacteria. The number of cells doubles as the generation time passes and the graph increases in a straight line. Stationary phase is when the growth rate of bacteria slows down and eventually stabilises. In the graph, stationary phase can be identified when the graph plateau. The death rate of the bacterial cell and the reproduction rate of bacterial cell will be the same during this phase. The deficient of nutrients and the excess waste materials will lead to destruction of many bacterial cells but not all of them. The last phase which is the death phase is when the nutrients in the culture is very low and the bacterial cells starts dying due to lack of nutrients. The death rate of the bacterial cell is higher than the reproduction rate of bacterial cell. From the growth curve, the death phase can be identified when the graph starts declining.

There are several environmental factors that will affect the bacteria growth for example temperature, nutrients requirement and pH concentration. Temperature is the most essential factor in the bacterial growth. The supply of oxygen is needed for the growth of the microbes. There are a few ways in measuring the microbial growth such as by studying the organisms growth rate in broth culture using optical density (Edwards et al., 2009). The optical density changes linearly with the bacterial cell concentration which is calibrated with the dry weight.

To study the growth of bacteria, the two important parameters are growth rate and the doubling time (Srivastava and Srivastava, 2003). The doubling time differs for different species of bacteria as the environmental factor plays a role in it as well such as the generation time for Mycobacterium tuberculosis is 360 mins however the generation time for Escherichia coli is 20 mins. The cell mass of the bacteria can be measured indirect and direct method. For the direct method, the bacteria dry weight is used however for the indirect method spectrophotometer is used to measure the turbidity (Madigan et al., 2009). Bacteria's are usually grown in a batch culture which is a closed system in laboratories. The bacteria culture is grown with one batch of medium throughout the whole incubation process and the bacterial growth population is studied (Willey et al., 2011).

Aims:

The aim of the experiment is to contrast the growth rate between the generation time of shaking E.coli culture and non-shaking E.coli culture. The four phases of the growth curve was identified if the bacterial cells are dividing as they are supposed to. The environmental factors that affects the bacteria growth is also identified.

Materials and Methods

The experiment was done according to MIC2011 Practical Lab Manual (2012) pages with the following changes:

2-3 hour of culture of E.coli was used instead of Vibrio natriegens in 12ml of NB medium instead of 30ml of BHI medium (+2.5% NaCl) in a mineral bottle instead of Erlenmeyer flask and incubated at 37℃. 200 ml of BHI medium was replaced with 200ml of NB mediuim in a 500ml Erlenmeyer flask. 100ml bottle containing 50ml of BHI medium was scraped off. Instead of 30ml bottle containing10ml of formalin, 9ml NB in mineral bottle was used. A blank spectrophotometer cuvette was prepared using 2ml of uninoculated nutrient broth instead of BHI and 1 drop of formalin was not added. The samples were diluted ten fold in nutrient broth medium instead of three fold in BHI medium when the optical density of sample is greater than 0.7.

Results

In a flask which has been pre warmed and shaking at 101,12 mL of E.coli culture was added into 200mL of nutrient broth. The flask was placed in a water bath at 37.5℃. 2mL of uninoculated nutrient broth was used as a blank and every time the optical density reading was taken the nutrient broth was used to blank. 2mL of E.coli culture was taken at every 10 minute interval to measure the turbidity by optical density using a spectrophotometer at 600nm wavelength. The result of the turbidity of the E.coli culture was taken till 140 minutes at every 10 minutes interval.

Figure 1: Graph of log of optical density of E.coli shaking culture at 10 minutes interval over time (minutes).

Figure 2: Graph of optical density of non-shaking E.coli culture at 10 minutes interval over time (minutes).

Calculations for mean growth rate

Shaking E.coli culture Non-shaking E.coli culture

n=log⁡〖Nt-logNo〗/0.301

n=log⁡〖0.674-log⁡0.010 〗/0.301

= ((-0.171)-(-2))/0.301

n= 6.074

Mean growth (µ) = n/t

= 6.074/120

= 0.05

n=log⁡〖Nt-logNo〗/0.301t

n=log⁡〖0.247-log⁡0.032 〗/0.3011

= ((-0.607)-(-1.495))/0.301

n= 2.950 generation/minute

Mean growth (µ) = n/t

= 2.950/100

= 0.03

Calculations of mean generation time

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