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This experiment is carried out to analyze the growing dynamicss of micro-organisms in shingle flask. E.coli is grown in a LB broth medium and being fermented for 24 hours. Throughout the agitation, the cell civilization is taken out for every 3 hours and protein trial, glucose trial and cell dry weight are being performed. As for the optical denseness analysis, the optical density reading from the spectrophotometer is taken while for the glucose trial, the reading of glucose degree is taken from the YSI 2700 Select Biochemical Analyzer or can besides being performed by utilizing DNS reagent and the optical density value is taken. These optical density values will so being compared with the standard curve to acquire the glucose concentration inside the shingle flask at peculiar clip. The cell dry weight, in the other manus, is taken after the mass concentration is being dried overnight in the oven. The weight of the viral which contains the biomass before and after the drying procedure is recorded to acquire the dry cell weight.

For the optical denseness of the cell, the optical density value showed an increase which bespeaking that the cell was turning and figure of cell is increased in the shingle flask. The glucose concentration, nevertheless, can non be determined as the optical density values were increased and decreased unevenly and comparing can non be made with the standard curve as the information for the standard curve are non consistent giving inaccurate curve. Therefore no decision can be made about the glucose concentration in the shingle flask. Purportedly, as the figure of cell increased, the glucose concentration would diminish as the glucose ingestion by the cells is increased.

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The dry cell weight in the other manus can be seen that there is an increase from the beginning of the cultivation until the 6th hr and showed unstable alterations until the 24th hr. Purportedly, as the figure of cell increased inside the shingle flask, the cell dry weight besides should be increased.

Introduction

Agitation can be carried out as batch, uninterrupted and fed-batch procedures. In this experiment, the shingle flask agitation is being used. Shake flask agitation is the illustration of batch agitation. In shingle flask, the civilization flask normally Erlenmeyer flask is being used to put and turning the micro-organism. It is the cheapest and easiest manner to civilization micro-organism aerobically, in little volumes of alimentary stock. It is a little graduated table equipment which equivalent to stirred armored combat vehicle bioreactor.

In order to forestall any taint to the civilization, agitate flask must be plugged. Different stopper can be made of cotton-wool, glass wool, polyurethane froth, gauze or man-made hempen stuff. The stopper has to forestall airborne micro-organism from acquiring into the medium while at the same clip leting free flow of air into the flask.

The civilizations are incubated at certain temperature and agitating frequence in an brooder shaker to accomplish a needed growing rate. The shaking agitates the medium and the civilization to maintain the mixture comparatively homogenous and besides to guarantee aeration, making an aerophilic status. In batch civilization, there is neither input supplied nor end product generated throughout the agitation. The average civilization is ab initio inoculated with the micro-organism. The growing keeps increasing until at certain extent, the growing is inhibited because of the diminishing substrate concentration and the presence of toxic metabolites.

Purposes

To analyze the growing dynamicss of micro-organism in shingle flask experiment

To build a growing curve including slowdown, log, stationary and decease stages

To find the Monod parametric quantities

Theory

Shake flask agitation is one of the illustrations of batch agitation. Batch civilization is an illustration of a closed civilization system which contains an initial, limited sum of food. The inoculated civilization will go through through a figure of stages. After an vaccination there is a period during which no growing appears to take topographic point. This period is referred as the slowdown stage and may be considered as a clip of version. In a commercial procedure, the length of the slowdown stage should be reduced every bit much as possible. Following a period during which the cell bit by bit increases, the cell grows at changeless, maximal rate and this period is known as the log stage or exponential stage. The exponential stage may be described by the equation below:

= µx — — — — — — — — — -1

where

ten is the concentration of microbic biomass

T is the clip, in hours

µ is the specific growing rate, in hr -1

on integrating, equation ( 1 ) gives

= — — — — — — — — — 2

where

is the original biomass concentration

is the biomass concentration after clip interval, T hours

During the exponential stage, the being is turning at its maximal specific growing rate, for the prevalent conditions.

Equation 2 predicts that growing will go on indefinitely. However, growing consequences in the ingestion of foods and the elimination of microbic merchandises. Therefore after a certain clip the cell growing rate will diminish until growing ceases. The surcease of growing may be due to the depletion of some indispensable food in the medium when there is restriction in substrate.

The lessening in growing rate and the surcease of growing due to the depletion of substrate may be described by the relationship between µ and the residuary growth-limiting substrate as follows:

µ =

where

= maximal growing rate

s = residuary substrate concentration

= substrate use invariable

Figure 4.1: The graph demoing the relationship between the parametric quantity of the Monod equation.

The stationary stage in batch civilization is the point where the growing rate has declined to zero. In the other word the growing rate is tantamount to the decease rate. The cell decease is might due to the alimentary restrictions due to their incorporation into cells during log-phase growing or a build-up of toxins due to their release of agitation merchandises besides during log-phase growing.

The decease stage is the consequence of the inability of the bacteriums to transport out farther reproduction as status in the medium become less and less supportive of cell division. The food is highly deficient for the growing of the micro-organism. Finally, the figure of feasible bacterial cells begins to worsen at an exponential rate. Industrial agitation is normally interrupted at the terminal of the exponential growing stage or before the decease stage begins.

Figure 4.1: Growth curve of micro-organism based on cell figure analysis

Apparatus and stuff

E.coli

Luria Bertani Broth

Distilled H2O

Shake flask

Cotton-plugged

Incubator shaker

Cuvettes

Centrifuges

Micropipetor

Pipette tips

Laminar flow

70 % ethyl alcohol

Lighter and Bunsen burner

Graduated cylinder

Schott bottle

DNS reagent

Procedures

Part 1: Preparation of inoculated agitation medium

500ml shingle flask, Bunsen burner, mensurating cylinder, LB broth and inoculants are brought into the laminar flow.

Under sterile technique, 50 milliliter of media is transferred into 500ml shingle flask.

Then 6 milliliter of inoculants is added into the shingle flask ensuing in concluding volume of 56ml.

The shingle flask is plugged with cotton-plugged.

The shingle flask is swabbed with 70 % ethyl alcohol.

The shingle flask is incubated at 350 revolutions per minute ; T=30EsC ; 24 hours.

Part 2: Sampling for cell dry weight

1ml of biomass concentration is taken out.

The 1ml biomass concentration is transferred into micro extractor tubing. An empty micro extractor tubing must be weighted foremost.

The sample is so centrifuged for 10 proceedingss at 10000 revolutions per minute.

After that, the supernatant of the sample is taken out carefully without taking out any biomass.

The biomass is so left dried inside an oven at 80C for nightlong.

The dried biomass is so being placed inside a dessicator to allow it chill before quickly weighing on an analytical balance.

Part 3: Glucose analysis

1ml of biomass concentration is taken out.

The 1ml biomass concentration is transferred into micro extractor tubing.

The sample is so put onto turntable of YSI 2700 Select Biochemical Analyzer for direct analysis of glucose concentration.

Another method of glucose analysis is by utilizing DNS reagent.

1.5ml of DNS reagent is added into 0.5ml of the biomass sample inside a capped trial tubing

The mixture is heated at 90EsC for 10 proceedingss to develop the reddish-brown coloring material.

The het mixture is so cooled to the room temperature for 2-3 proceedingss in a cold or ice H2O.

The mixture is so being diluted with 10ml of distilled H2O.

The optical density is checked with a spectrophotometer.

Part 3: Sampling for optical density analysis/ optical denseness

2ml of biomass concentration is taken out and being transferred into micro extractor tubing.

The spectrophotometer is calibrated to zero by clean consisting of 2ml LB Broth.

The biomass concentration is so being transferred into a cuvette and optical denseness measuring is taken with wavelength set at 600nm.

More optical density means higher figure of cell.

Part 4: The readying of glucose criterion curve

The 20g/L, 40g/L, 60g/L, 80g/L and 100g/L of glucose concentration is prepared by weighing the suited sum of glucose and diluted with 10ml of distilled H2O.

1.5ml of DNS reagent is added with 0.5ml of the glucose sample inside a capped trial tubing

The mixture is heated at 90EsC for 10 proceedingss to develop the reddish-brown coloring material.

The het mixture is so cooled to the room temperature for 2-3 proceedingss in a cold or ice H2O.

The mixture is so being diluted with 10ml of distilled H2O.

The optical density is checked with a spectrophotometer

Consequences

Table 7.1: The consequences demoing the optical denseness of the cell, cell dry weight and glucose concentration per clip

Hour

Optical density readings: Optical denseness ( spectrometer wavelength: 600nm )

Biomass concentration: cell dry weight after 24h ( g )

Glucose concentration ( g/L )

Initial

Concluding

Internet

optical density

Glucose

0

0.340

1.052

1.066

0.014

0.025

3

1.812

1.153

1.170

0.017

0.071

6

2.597

1.045

1.070

0.025

0.066

9

2.688

1.153

1.175

0.022

0.088

12

2.734

1.167

1.183

0.016

0.079

21

2.801

1.157

1.176

0.019

0.094

24

2.800

1.156

1.175

0.019

0.087

Figure 7.1: The graph demoing the optical density value for optical denseness

Figure 7.2: The graph demoing the cell dry weight per clip

Table 7.2: Datas for standard curve of glucose trial

Glucose concentration ( g/l )

Optical density

0

0.039

20

2.141

40

2.750

60

2.760

80

2.505

100

1.791

Figure 7.3: Graph demoing the standard curve for the glucose concentration

Calculations

Cell dry weight

Net weight = concluding weight – initial weight

= 1.066g – 1.052g

= 0.014g

Discussions

This experiment is carried out to analyze the kinetic growing of micro-organism. E.coli is selected as the cell and being cultivated inside a shingle flask. The growing of micro-organism in shingle flask is a simple method of agitation. The foods for the micro-organism are being supplied by the media which contain the C beginnings. The flask is shaken during the cultivation to blend the cell and the media ; increase the homogeneousness between these two and besides to supply aeration for the cells. The civilization is gone through the agitation procedure for 24 hours. Within that period, the biomass/cell sample is taken out for every 3 hours to analyse the concentration of the cell ( g/L ) , the cell dry weight and the glucose concentration.

In order to analyse the concentration of the cell inside the flask, optical density reading for the optical denseness is taken from the spectrophotometer. The higher the optical density reading means higher figure of cell presence inside the flask at a peculiar clip. As for this experiment, the optical density reading is increase from the beginning of the experiment until the 21st hr and lessening somewhat at the 24th hr. It can be explained that the figure of cell addition throughout the cultivation bespeaking that the cell is turning. In the other manus, the lessening in cell figure in 24th hr indicating that the cell growing has reach its slowing stage where the growing of the cell is started to decelerate down. The slowing growing stage is where the civilization is in a transeunt province. During this phase there are feed/back mechanisms that regulate the bacterial enzymes involved in cardinal metabolic stairss to enable the bacteriums to defy famishment. There is much turnover of protein for the civilization to get by with this period of low substrate handiness. In cell growing, the cell will travel through several stages like slowdown, exponential, slowing, stationary and decease stage.

In cell cultivation, the cells themselves need nutrient or C beginnings like glucose for growing. In batch agitation for illustration in this experiment, the glucose can be the confining factor for the cell growing or we called it as substrate modification growing. For this status, the Monod equation can be used to foretell the growing rate and the cell concentration inside the shingle flask. In add-on, the glucose concentration can be known by proving the cell sample into the glucose analyser and the direct glucose concentration can be obtained. In other manner, the glucose concentration is besides being obtained by blending the sample with DNS reagent. The DNS reagent will be reduced to 3-amino,5-nitrosalicylic acid in the presence of free carboxyl group ( glucose ) and optical density reading can be taken through the spectrophotometer.

As for this experiment, the glucose trial showed no form of alterations in optical density values. These values addition and lessening unevenly. This might be due to some errors occurred during the glucose trial where the volume of sample and DNS reagent that need to be assorted is falsely taken. This has affected the truth of the optical density reading. From the optical density reading, the concentration of the glucose can be obtained by mentioning to the glucose criterion curve. The glucose concentration should be decreased as the figure of cell inside the flask is increased. This is because as the figure of being additions, foods are consumed and going lesser. However, this can non be shown from the consequences obtained due to some errors occurred throughout the experiment.

Figure 9.1: The alterations in sum of glucose, lactose and biomass in cell civilization per clip.

Another analysis that can be performed to analyse the cell sample is by taking the dry weight of the cell. In this method, the cell is being taken out from cultivation flask and transferred into viral tubing. The tubing is the being centrifuged to divide the supernatant with the cell. The remained cell is so being dried inside an oven for 24 hours. The dry cell weight is eventually taken to cognize the weight of the cell that nowadays at peculiar clip during the cultivation. In this experiment, the cell dry weight is increased from 0th hr until 6th hr and bit by bit decreased from the 9th hr to 12th hr and increased until the 24th hr. The cell dry weight should increase when the figure of cell increased inside the shingle flask.

Decisions

At the terminal of this experiment, micro-organism is suited to be fermented inside a shingle flask and it is a simple method to look into the growing dynamicss of the micro-organism. Knowledge of microbic growing dynamicss is indispensable to find when to reap the civilization for different intents. For a growth-linked merchandise, it is desirable to reap the civilization at the late exponential growing stage. On the other manus, for a non-growth-linked merchandise, it would be desirable to reap the civilization at the stationary growing stage.

As micro-organism will travel through several stages in their growing, several analyses on the cell demand to be done to cognize the growing dynamicss of the cell and the continuance for each stage. This includes the cell concentration, glucose concentration and besides the cell dry weight analyses. This method can be done in the research lab before the agitation or the cultivation of bugs in big graduated table is performed. Growth dynamicss trades with the rate of cell growing and how it is affected by assorted chemical and physical conditions. During the class of growing, the cells is continuously altering and accommodating itself in the media environment, which is besides continuously altering in physical and chemical conditions.

In decision, the microbic civilization in batch civilization system ( agitate flask system ) goes through a slowdown stage, exponential growing stage, slowing growing stage, stationary stage and sometimes the decease stage depends on the terminal merchandise desired. The substrate concentration in the civilization medium and growing parametric quantities, such as glucose concentration alterations correspondingly throughout the growing phases. Therefore, the physiology of the micro-organism is ever in a transient phase, subjected to a continually changing civilization conditions. Consequently, merchandise formation is confined to a certain period of cultivation, for illustration antibiotics would merely be produced in the decelerating and stationary growing stages.

The batch civilization system is still widely used in certain industrial procedures for illustration brewery industry because of its easy direction of provender stocks. These advantages allow the usage of unskilled labors and low hazard of fiscal loss. Low degree of microbic taint in fermented merchandises is at clip tolerable, every bit long as the microbic contaminations are non infective and make non change the coveted belongingss of the merchandise, such as gustatory sensation, coloring material and texture.

Recommendations

Aseptic technique must be practised when managing biomass concentration to avoid any taint.

Cuvette must be wiped flawlessly to forestall any abrasion that would impact the spectrophotometer reading during protein trial.

This experiment must be carried out under the laminar flow to forestall any taint to the civilization.

The supernatant of cell concentration should be taken out carefully without any taking out of the biomass.

The cap of the viral must be opened to fix the drying procedure of the biomass in the oven.

Wash manus after managing the civilization.

Disinfect the work country with 70 % intoxicant before managing the civilization.

Dispose of all contaminated stuffs in appropriate containers.

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