Shake Flask Fermentation

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Table of Contents
1.0 Abstract2
2.0 Introduction2
3.0 Aims3
4.0 Theory3
5.0 Apparatus5
6.0 Procedure5
7.0 Result7
8.0 Calculation10
9.0 Discussion11
10.0 Conclusion13
11.0 Recommendation13
12.0 References13
13.0 Appendix14

1.0 Abstract
In this experiment, Escherichia coli is used as a sample to study the growth kinetic of microorganism in shake flask. A different volume of E.coli was transferred into 250ml Erlenmeyer/shake flask containing media for the nutrient of microorganism. The different volume of microorganism transferred will give the different effect of reading on the constant volume of media used. There are three ways to test the growth kinetic rate of microorganism on shake flask, which are by optical density (OD), glucose analysis and cell dry weight (CDW). Then, from these test the result then used to plot the growth curve graph which next use to compared and analyze the growth curve of microorganism.

2.0 Introduction
Shake flask fermentation is one of the fermentation method which are widely used for screening of high producing strains. The shake flask fermentation is the simplest way to do the fermentation using small amount volume of nutrient broth in laboratory. A nutritionally rich medium, Lysogeny broth or luria bertani (LB) is used for the growth of bacteria. Usually the shake flask used is between 250ml to 500ml range. From the previous research, it is shown that smaller volume of shake flask give better oxygen transfer rate but it is only suitable for short term fermentation, otherwise the medium will be evaporate. The shake flask have several design which are standard shake flask or Erlenmeyer flask, flying saucer shake flask, shake flask with baffles and flat bed Thompson bottle. In this experiment, the standard shake flask is used.

Erlenmeyer flask which equipped with cotton wool stoppers and autoclaved including the nutrient broth inside the flask. Then, the flask is allowed to cool to the room temperature before some microbes are allowed to grown inside the flask and put into the shaker machine. Most of the shaker machine are suitable for flask fermentation , although there are shaker which can be adjustable to allow the use of Other important component in shake flask is plugs. There are different types of plugs that can be used s such as plugs made from cotton-wool, glass wool, polyurethane foam or synthetic fibrous material. The function of plugs is to prevent airborne microorganism from getting to the medium while at the same allowing free flow of air into the flask. Shaker machine is designed to assist with oxygen transfer for aerobic respiration microorganism. For more precisely defined environment, incubator shaking cabinet can be very well use. These cabinets can control the temperature, illumination, gaseous levels and humidity. The movement of shaker normally rotary shaking action or reciprocating shaking action is produced. And by increasing the speed of a shaker can increase the oxygen transfer rate of a particular flask. 3.0 Aims

1. To study the groth kinetics if microorganism in shake flask experiment. 2. To construct a growth curve including lag, log, stationary and death phases. 3. To determine the Monod parameters.

4.0 Theory
As the experiment required safety handling and carefully conduct, growth culture in a shake flask will be done through several techniques. The preparation of culture media should be done under laminar flow safety cabinet and the transfer of culture to media and from culture media to cuvette must undergo aseptic technique first to avoid unwanted microorganism enter the culture media. Measuring the sample optical density to develop a growth curve should be done by using spectrophotometer. The step involve is quick and easy. For sample containing E.coli, the particular wavelength should be set to 600nm. Each reading should be recorded. As the microorganism grow with time, a growth curve can be used...
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