Preparation Of Media And Growth Of Bacteria Biology Essay

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To grow and reproduce, microorganisms require nutrients as their source of energy and certain environmental conditions. Adaptation to different habitats has been acquired by microbes living in the exposed environment. In the laboratory, however, these requirements are to be met by a culture medium. Therefore, media preparation is an essential aspect in microbial growth.

There is a wide variety of media which can be used for laboratory purposes. Generally, similar procedures can be used in the preparation of media. In this practical, YT broth is prepared from yeast extract, trypton, NaCl and agar. YT broth is a commonly used bacterial Esherichia coli culture media in molecular biology. Usually, YT medium is applicable for phage DNA production. Compared to LB broth, YT is a richer medium in which it supports higher cell density and a longer growth period for E. coli.

Bacteria are the most abundant and most important biological component as they involve in the transformation and mineralization of organic matter in the biosphere (Cho & Adam, 1988; Pomeroy & Wiebe, 2001). Generally, growth is defined as an increase in number of cells. Bacterial cell growth depends upon a large number of cellular reactions such as transforming energy. Syntheses of small molecules are performed to build macromolecules and to provide various cofactors and coenzymes for biochemical reactions. Polymerization of monomers builds the macromolecules in the bacterial cell. As the macromolecules accumulate in the cell, they are assembled into new structures with specific functions such as the cell wall, cytoplasmic membranes, ribosomes and flagella, if to name a few, and eventually lead to cell division.

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E. coli is used as a test organism in this practical for investigating the bacterial growth. Such a growing rod shaped cell bacterium elongates to approximately twice their original length and then forms a partition that constricts the cell into two daughter cells. This process is known as binary fission. A partition called the septum is formed by the inward growth of the cytoplasmic membrane and the cell wall from opposite directions. The septum continues to grow inwards until the two daughter cells are pinched off. When one cell separates from to form two daughter cells, one generation has occurred and the time required for binary fission is known as the generation time. Under the best nutritional conditions, the generation time of a laboratory E. coli culture is approximately 20 minutes (Buettner et al., 1973).

The growth stages of bacterial cells are described in a growth curve consisting of the lag phase, exponential phase, stationary phase and death phase (Zwietering et al., 1990). In the lag phase, the bacterial cell begins to grow only after a period of time due to new environment being inoculated. The time interval of lag phase depends on the growth conditions and the history of the inoculum. If the growing culture is transferred into the same medium under the same conditions, there is no lag phase and exponential phase starts immediately. However, if the inoculum transferred is taken from the stationary phase in an old culture, lag phase is observed. This is because certain essentials of the cell have depleted and time is required for biosynthesis of new constituents. The lag phase is also observed when the cells are transferred from a rich culture medium to a poorer culture medium. In the poorer condition, the cells need to synthesize the essential metabolites that are not present in the previous medium. Thus, time is needed by the cells to produce new enzymes. In the exponential phase, the cell divides to form two cells, both the total cell number and mass double but there is no change in the average cell mass. The cells are said to be in their healthiest state. The rate of exponential growth varies widely among microorganisms. The rate is influenced by environmental conditions and the genetic characteristics of the organism itself. In the stationary phase, the essential nutrients of the culture medium are used up and at the same time, the wastes produced by the bacterial cells accumulate in the culture medium. Thus, growth is inhibited. The exponential growth is ceased and the cells reach the stationary phase. There is no net increase or decrease in the cell number and hence the growth rate of the cells is zero. The cells enter the death phase of the growth cycle after reaching the stationary phase whereby cell lysis occurs.

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Figure 1: Typical growth curve of a bacterial population.

The growth of E.coli is investigated under various cultural conditions, including effect of pH, temperature and aeration). Effect of pH is ascertained by preparing media of different pH values. Temperature is always a potentially limiting factor by affecting all chemical and biochemical processes and thus is viewed as an interactive factor (Pomeroy & Wiebe, 2001). Aeration is done by shaking so that air space volume can be increased. As such, good and excellent aeration is stimulated.

MATERIALS

Media components (yeast extract, trypton, NaCl and agar), E. coli culture, 20x petri dish, 5x250 ml conical flask and 1x100 ml conical flask, plus cotton wool plugs in aluminium foil, Whatman paper

METHOD

Media Preparation

The five 250 ml conical flasks and one 100 ml conical flasks were prepared and labelled as M1-M10 on the first day of practical.

100 ml of YT broth was prepared in 250 ml flasks, with the composition as trypton 1.6%(w:v), yeast extract 1%(w:v), and NaCl 0.5%(w:v).

The pH was adjusted to those shown in table 2.1, followed by autoclaving.

500 ml agar was prepared in 1L flask, in which composed of YT broth and agar (13.5 g/L).

The prepared medium was autoclaved.

The agar was poured on 20 petri dishes.

Inoculation

Day 2 was begun with inoculating M1 to M8 with 1.0ml E. coli, while M9 and M10 as control set.

Incubation was performed at 37°C for 24 hours.

Whatman paper was dried overnight.

Analysis

Prior to an analysis, the culture was well shaken and the sampling was done under aseptic condition.

Measurement of cell density and variable cell (as colony forming units, cfu)

After the overnight incubation, a 3.0ml aliquot of each culture was taken and the absorbance was measured at 660nm.

M9 and M10 was used as the blank.

Data obtained was tabulated.

Measurement of final pH of broth

The final pH of the cell culture was measured.

The results were also recorded in table 2.1.

Measurement of CFU

At day 3, a serial dilution was done from 10-2, 10-4, 10-6, 10-8 to 10-10.

Hockey stick spreading technique was performed to plate out the sample. Two replicates were done for each plate, labelled 'a' and 'b' respectively.

The culture was incubated at 37°C for 24 hours.

The changes were observed and recorded on the next day, and thereby determining the CFU, as what recorded in table 2.2(a) and 2.2(b).

Measurement of biomass

The Whatman paper dried was weighed and the initial weight was recorded.

A 10ml aliquot of the content of the flasks was filtered using the Whatman paper by washing with distilled water.

The paper together with the content was dried in oven at 70°C overnight.

On the next day, the final weight was measured and thereby obtaining the net weight gain of the filter paper.

The result was recorded in table 2.3.

RESULTS

The pH change and the absorbance values measured were recorded in table 2.1.

Flask

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

Pre-incubation pH

4.0

7.0

10.0

7.0

4.0

7.0

10.0

7.0

7.0

7.0

Post-incubation pH

3.96

7.08

10.16

7.16

4.13

7.21

9.93

7.73

7.12

7.14

OD660nm

0.108

1.267

0.01

0.916

0.191

1.906

0.056

1.812

0.0

0.0

Growth condition

No shaking (with air space)

No shaking (without air space)

Shaking at 200 rpm (with air space)

Shaking at 200 rpm (without air space)

-

Table 2.1: pH change and absorbance measured for M1 to M10

CFU for plate labelled 'a' and for plate 'b' was calculated using the formula below, which was then recorded in table 2.2(a) and 2.2(b) respectively.

CFU = colony number x dilution factor

= x cfu/0.1ml

= ? cfu/ml

Table 2.2(a): CFU for plate labelled 'a'

Flask

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

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Dilution (10-4)

55

22

0

>300

1

161

0

>300

0

0

CFU/ml

5500000

2200000

0

TMTC

100000

16100000

0

TMTC

0

0

Dilution (10-6)

0

13

0

185

0

24

0

3

0

0

CFU/ml

0

130000000

0

1850000000

0

240000000

0

30000000

0

0

Flask

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

Dilution (10-4)

13

>300

0

>300

0

>300

0

>300

0

0

CFU/ml

1300000

TMTC

0

TMTC

0

TMTC

0

TMTC

0

0

Dilution (10-6)

0

3

0

89

0

32

0

4

0

0

CFU/ml

0

30000000

0

890000000

0

320000000

0

40000000

0

0

Table 2.2(b): CFU for plate labelled 'b'

The biomass was calculated using the formula below and was recorded in table 2.3.

Biomass (g/ml) =

Flask

M1

M2

M3

M4

M5

M6

M7

M8

M9

M10

Initial weight of filter paper

1.14

1.16

1.14

1.17

1.19

1.15

1.16

1.19

1.16

1.17

Dried weight of filter paper

1.15

1.17

1.14

1.17

1.20

1.17

1.16

1.19

1.16

1.17

Mass Difference

0.01

0.01

0.0

0.0

0.01

0.02

0.0

0.0

0.0

0.0

Biomass (mg /ml)

0.001

0.001

0.0

0.0

0.001

0.002

0.0

0.0

0.0

0.0

Table 2.3: Biomass

DISCUSSION

From the tables in the result section and observation, we analyze the growth of E. coli under various pH and aeration condition. Measuring OD and calculating CFU give relevant and supportive information to our experiment. However, the biomass calculated is not applicable to accurately ascertain the factors affecting the growth as some errors occurred during the measurement of biomass, probably.

Cell density and variable cell

OD stands for optical density which indicates the wavelength of light. In this practical, OD660 measures the light absorbance by E. coli cultures, which correspond to the E. coli cell density in a given volume. OD660 determines whether the cells are ready for making cell stocks or as a competent cell. In other words, it can be said that in this part, OD660 was used to ascertain the quantity of E. coli grown in the YT broth after incubating overnight at 37°C. Flasks M1 to M8 were inoculated with 1.0 ml of E. coli culture. The result showed that growth had occurred. On the other hand, M9 and M10 served as the control set in which inoculation of bacteria was not applied, in addition to acting as blank during analysis with spectrophotometer. From table 2.1, it was shown that the OD of both M9 and M10 appeared to be zero. This shows that there was no sign of growth of E. coli in the two flasks.

Spreading of plates was done in class II laminar flow cabinet. Sterile operation in cell culture is vital where it can affect the CFU of E. coli. This minimizes the chance of culture contamination. On top of that, the safety of the operator is ensured (Betler, 2004). From the aspect of CFU, as what shown in table 2.2(a) and 2.2(b), there was abundant of cells in M2, M4, M6 and M8. The cell density was still high even with the dilution of 10-6. Therefore, it was determined that E. coli has the optimum growth pH at 7.0.

Effect of broth's pH on growth

Different growth condition gives different OD values. Optimum pH of E.coli growing in a culture at 37oC is around 7.0. E.coli is not able to tolerate under extremely alkaline and acidic environments because most of the enzymes found in the bacterium are very pH-sensitive. These enzymes carry out the important processes in E.coli. When there is extreme change in pH, enzymes in E.coli become denatured. Denature of enzymes in E.coli can lead to all sorts of interruptions to biochemical processes or even causes death of the E.coli. From table 2.1, it was found that there was no growth of E.coli in both flask M3 and M7 with pH 10.0; as the absorbance measured were 0.01 and 0.056 respectively. Whereas, there were some E.coli grow in flask M1 and M5 with pH 4.0, which have OD recorded 0.108 and 0.191 respectively. Thus, it can be concluded that E.coli seems to be more tolerate to lower pH (acidic condition) than higher pH (alkaline condition).

Since pH 7.0 is the optimum pH for the growth of E.coli at 37oC, there were many colony of E.coli growing on the agar plates which spread with the sample from flask M2, M4, M6 and M8.

There are some changes in the pH before and after the incubation. This is because there is formation of inhibitory products such as organic acids during the growth of E.coli in the YT nutrient broth.

Effect of aeration on growth

pH of medium is determined by medium composition, buffers, cellular metabolism and aeration. NaCl is used as buffer, where the depletion of this organic buffer will cause an E. coli reaches its maximum pH limit. Hence, care has to be taken to balance the organic buffer, sugar content and aeration medium because low aeration leads to production of acids; High aeration causes cells to use organic acids as carbon source, and thus increases the pH of medium. In brief, selected aeration also helps in maintaining cell's pH.

Theoretically, shake flask incubator reduces the solid and liquid inter-phase, thus minimizing the mass transfer. Increased speed and momentum cause cells to lyse among themselves. Increased speed also increases the formation of foam, thus trapping gas and resulting less gas in the liquid. Since cells require nutrients for survival, absence of gas as nutrient source will cause cells to die. Hence, shaking flask actually plays an important role in bringing an improvement to mass transfer between the gas phase outside the shake flask and the liquid phase inside the shake flask. Shake flask incubator can provide a suitable environment by ensuring sufficient transfer of oxygen gas to the cell culture by appropriate mixing.

Agitation will increase the aeration of the bacterial growth of E.coli. This is because oxygen is important for high density growth of E.coli cells. Agitation is controlled by the shaking speed of a shaker incubator. Thus, from the observation, Whatman paper with content from M6 has bigger biomass than others. The biomass of M6 is 1.0 mg/ml. This is because flask M6 has been shaken at 200rpm and the flask contains air space which helps in the growth of E.coli cells. Therefore, the media without shaking or without air space or without both of these growth conditions has less growth of E.coli. Thus, M6 has the best growth condition for E.coli if compared with other media.

However, from the observation in this experiment, there is more E.coli colony grows in M8 (growth condition with shaking at 200rpm but without air space) than M6 (growth condition with both shaking at 200rpm and with air space) in the culture with serial dilution 10-4. Theoretically, there should be less E.coli colony grows on the plate which spread with the culture from M8 since flask M8 has less flavour growth condition for the growth of E.coli. This unexpected observation may be because the sample with serial dilution 10-4 is not well - shaken before take 1.0ml of aliquot to spread on the plate.

From our result, it was again determined that the growth rate of E. coli is not affected by shaking. This is proven through our experiment where there was no significant difference in yields with or without shaking cultures. When air is supplied, however, shaking cultures shows a higher yield than static cultures. The main purpose of shaking cultures at 20 rpm is to mix thoroughly the nutrient concentration in broth, with constant temperature, 37°C. 37°C is the optimum growth temperature for E.coli. Shaking prevents cells localizing at the bottom of the broth which causes a faster depletion of nutrient as time passes. Nutrients still present in the broth but cells may not accessible to them. Hence, shaking minimizes areas of high and low nutrient concentration. The amplitude of the vibration controls the intensity of fluid mixing. Shaking broth brings advantage to non-motile cells because it enables the metabolism rate to continue, in addition to benefiting mobile cells by reducing energy on mobility. Besides, shaking increases the aeration of broth which leads to a higher yield of E. coli.

From the OD values, OD at pH 7 shows the highest value for both shaking and non-shaking media because pH 7 is the most suitable medium for growth of E.coli. However, pH 7 from shaking broth has a comparatively higher OD reading compare to non-shaking broth due to shaking enhances the growth of E.coli. However, OD of pH 4 is far higher than OD of pH 10 because E.coli can still grow on pH 4, but pH 10 does not really support growth of E.coli. As for M9 and M10, both broths do not show any OD reading value because no E.coli culture is added into both broths. When pH and vibration are held constant (i.e. pH 7 with shaking), broth with aeration shows a higher OD value compare to non-aeration. This means that a higher E.coli cell density in aerated broth compare to non-aerated broth.

When other factors are held constant, air space does not significantly affect E.coli yields, because E.coli is a facultative anaerobe, which means E.coli is able to produce energy during aerobic respiration and switch to anaerobic respiration when oxygen is depleting in the environment. Thus, E.coli grows in both with or without air space broth. Supposedly, colony forming unit (CFU) in plates from medium with air space has a higher growth amount compare to medium with no air-space. This is because E.coli cells produce a large quantity of acetic acid with no air-space, and thus reducing the pH of broth and does not favour the growth of E.coli. Acetic acid inhibits growth condition under anaerobic growth condition. However, according to our result, when the pH and vibration are held constant (both of pH 7 and with no shaking), CFU is greater in non air space compare to with air space. This may be due to errors occur where the mouth of broth is not tightly covered which leads to entrance of oxygen molecules into the broth.

CONCLUSION

Sterile medium such as sterile YT broth is essential for bacteria growth. The optimum pH for the growth of E. coli is 7.0. Excellent aeration promotes growth of bacteria to a further extent than what good aeration does.

QUESTIONS

Write a flow chart for the above protocol before you start work.

Label all the conical flasks from M1 to M10

Add 100ml YT broth to flasks M1 to M3 and M5 to M7

Adjust the pH of the media to 4.0,7.0 and 10.0 using either HCL or NaOH

Autoclave and allow to cool to room temperature

For flask M4 to M8, autoclave 200ml of the medium in a bigger container and then pour the cooled medium into the sterile 100ml conical flask

Inoculate flasks M1 to M8 with 1.0ml of E.coli culture under aseptic conditions

Incubate with or without shaking as indicated from the table

Prepare M9 and M10 under the same conditions as for flask M2 and M6, but do not add any inoculum

Prepare and autoclave the agar

After an overnight incubation,take 3.0ml aliquot of each culture and measure the absorbance at 660nm

Do a serial dilution from 10-1 to 10-5 in a sterile microfuge tube and plate out 0.1ml of the sample at 10-4 to 10-5 in YT agar media

Incubate at 370C overnight

Determine the cfu/ml of the original culture and tabulate data

Measure the final pH of the cell culture and tabulate results

Filter 10ml aliquot of the content of the flasks using pre-weighted Whatman paper dried in an oven at 800C overnight

Measure the net weight gain of the filter paper as an indication of biomass

Tabulate results

Discuss your results with respect to the effect of pH of the media and aeration on bacterial growth and reasons for your observations.

In addition to nutrients, the pH of the growth medium is also important for E.coli growth rate and cell density. The optimal growth pH for E.coli is near neutral. E.coli cells can grow reasonably well over a range of three pH units (from pH 5.5 to 8.5). Extreme pH beyond this range will significantly decrease the cell growth rate and may sometimes even cause cell death. The minimum and maximum growth pHs for E.coli are pH 4.4 and 9.0 respectively. E.coli cells appear to tolerate a more neutral pH better than a high pH. This is clearly shown as E.coli grows well at pH 7 as shown on plate M2, M4,M6, and M8.In fact, extended exposure of E.coli cells to a high pH causes cell lysis. This is the reason why no colonies were found on plate M3 and M7. At the saturation or stationary phase, the pH of the E.coli culture in commonly used media is near its pH limits. pH is another limiting factor for cell growth in addition to nutrition exhaustion and accumulation of toxic metabolites. E.coli cells can also use sugars such as glycerol and glucose as carbon or energy sources. When the E.coli cells use these sugars as carbon sources, they will produce acetic acid and therefore lower the medium pH. Carefully balancing the sugar contents, and aeration conditions can maintain the culture medium pH near E.coli optimum growth pH or within the range of the three pH units. Low aeration conditions lead the cells to produce acids. High aeration conditions allow the cells to use organic acids as carbon source and increase medium pH. Selected aeration conditions can also help cells maintain its medium pH.

Do your data on absorbance at 660nm correlate with the cfu/ml and biomass data? Explain.

The data on the absorbance at 660nm correlates with cfu/ml as a lower OD660nm gives a high cfu/ml and vice versa. But the data of absorbance does not correlate with the biomass. Despite the inherent inaccuracy of the method, if the procedure is adequately controlled and calibrated the estimation of microbial numbers by optical density should be sufficiently accurate for use in preparing inoculum for cfu/ml testing.

Would you expect to obtain the same data if you were to scale up the experiment using a 1000L fermenter? Explain your answer.

No, as the size and volume of the fermentor increases, the volume and density of the E.coli culture will increase too so the CFU/ml will also increase.