Environmental Factors Affecting Growth of Bacteria II


Aim:
The aim of this experiment is to observe lethal effects of temperature, heavy metals and UV light on E.coli cells for obtaining how microbial growth can be controlled; and determine thermal death point and thermal death time.
Hypothesis in this experiment is to determine thermal death time by groups studied at different temperatures to figure out thermal death point and to expect to see increase at proper temperature range and also decrease out of optimum temperature range ; to determine increase or decrease according to each different heavy metals and lastly UV light decreases bacterial growth.
Introduction:
Lethal effects of temperature,
In organisms, organized structure and units are so important for optimum workplace. High temperature causes structures and organizations. Especially, enzymes firstly are affected. Their dimensional structures are broken and then they cannot work properly. By increasing temperature, membrane dispersed. Eventually, cells start to die. (1)
Thermal death point refers to a certain temperature to kill organism in 10 minutes. Thermal death time is a term for using to refer the time that how long it takes to kill an organism at a certain temperature. These terms are used often for these kinds of microbial growth researches. (2)
Oligodynamic action,
Oligodynamic action refers to effects of heavy metals to organisms in low concentrations. Heavy metals can attach enzyme structure and they can affect their activity easily. Many of them have dangerous impact for living organisms because of them. Some of them are also important with benefits. Some of them can join structure or take a place on enzymes as a cofactor like Mg+2 joining to photosynthesis. Each heavy metal has different effectiveness according to kind of organism. (3)
Effect of UV light on microbial growth,
UV light can pass and give damage cells by its own high energy-wavelength. Mostly, lethal effect is to DNA. DNA structure can change. Especially changes in genes on DNA are so essential for organism. Generally this change is a dimerization. Close thymine nucleotides can form dimer structure between each other. The dimerization is to prevent transcription and cells cannot produce proteins they need. Some essential limits of UV light are necessary to damage. Exposure time and intensity are some of them. Also sporulation is important. Bacteria that can produce spores can resist against UV light than the others. (4)
Material and Methods:
Materials:
E. coli culture
Heavy metal solutions (0,5M Cr3+ , Cr6+ , Mn2+ , Ni2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , Zn2+ , boric acid , Mg2+ and 0,05M Cd2+ )
Heater
Beaker
Bunsen Burner
Drigalski spatel
Ethanol
Agar media plates
UV source

Methods:
Lethal effects of temperature,
For determining thermal death point and thermal death time, each group chose a certain temperature (50, 60, 70, 80 and 900C) and each group measure by using same time interval (10 min) and using same steps. First of all, a portion of water was put into the beaker and it was put onto heater arranged to 800C, the degree of temperature our group used. After that, five agar plates were prepared. A thermometer was placed into beaker to check temperature. Before the time measurement, 100µl of E.coli was inoculated to agar plate for control group by spreading with Drigalski spatel and then bacteria were aseptically transferred to the tube immediately before being obtained in 80 degree warm water. The tube was put into 80 degree warm water and it was waited for 10 min. After 10 min, the tube was taken from beaker and 100µl of the sample in the tube was inoculated to agar plate by spreading with Drigalski spatel aseptically. This process was repeated for 40 minutes at 10 minute intervals and at each step, the plates were labelled with date, degree of temperature, name of group. The plates were incubated for one day at 370C. Next day, agar plates were observed and the results were recorded.

Oligodynamic action,
At this point of experiment, each group used two different heavy metals of 0,5M Cr3+ , Cr6+ , Mn2+ , Ni2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , boric acid , Mg2+ and 0,05M Cd2 for this step.
0,1 ml of E.coli culture was inoculated by spreading with Drigalski spatel for each two agar plates near flame. 0.2 ml from Fe3+, Cu2+ ; the heavy metals of our group. 20µl of Fe3+ was poured onto the middle of plate and then same process was done with 20µl of Cu2+ to the other plate. The two plates were labelled with date, name of heavy metals. The plates were left in incubator at 370C. Next day, diameters of circles heavy metals formed on plate by ruler was measured and recorded.
Effect of UV light on microbial growth,
Each group determined effects of UV light with different time intervals (30 sec, 1 min, 5 min, 10 min, and 15 min). Each group prepared one agar plate with E.coli sample by using aseptic techniques. Then, sample was waited according to time interval under UV light. Lastly, observation of effects of UV light was observed and recorded.
Results:
500C 600C 700C 800C 900C
Control +++ +++ +++ +++ +++
10 min +++ +++ +++ +++ –
20 min +++ – + + –
30 min ++ ++ – – –
40 min +++ ++ – – –
Table 1- the results of observation of lethal effect of temperature to determine thermal death point and thermal death time. (-: no growth, +++ : high growth, ++ : normal growth, + : low growth) after incubating at 370C.

Heavy metal solutions: Cr3+ Cr6+ Mn2+ Co2+ Cu2+
Diameter: 2.5 cm 1.5 cm 2.8 cm 3.3 cm 2.5 cm
Heavy metal solutions: Fe2+ Fe3+ boric acid Mg2+ Cd2+
Diameter: 0.4 cm 1.5 cm – – 3.5 cm
Table 2-The results of diameter of circles on plate after heavy metal effects (-: no effect, +++ : high growth, ++ : normal growth, + : low growth) after incubating at 370C.

UV exposure time Growth rate
30 sec +++
1 min +++
5 min +++
10 min +++
15 min +++
Table 3- The results of UV exposure effect by time according to comparison. (-: no effect, +++ : high growth, ++ : normal growth, + : low growth) after incubating at 370C.
Discussion:
The aim of this experiment is to determine effects and exposure of UV light, heavy metals and also thermal death point and thermal death time to know how the bacterial growth can be controlled.
Lethal effects of temperature,
At this step of the experiment, each group aimed to observe effects of a certain temperature to bacterial growth according to time. At 50, 60, 70, 80 and 900C, observations were done (Table1).
At 500C, bacterial growth is as expected. E.coli is a mesophilic bacterium (5). It means they can grow at not too hot or cold environment. 500C is so proper condition for E.coli bacteria. In Table 1, only at 30 min, a small reducing is seen. This small reducing may have occurred because of sudden heat change between beaker and spreading onto plate. At 600C, until 20 min, there is high growth as expected. Sudden heat change and out of optimum conditions may have caused a going down at growth rate. At 20 min, no growth may have formed because of experimental error while transferring or waiting for a long time or not keeping the temperature within a certain range or experimenter may have forgotten cooling spatula and bacteria may have died because of that. At 700C, within 20 min and after 20 min, decrease can be seen. Effect of high temperature on bacteria shows itself by time. At 800C, also same results are seen with 700C. Bacteria can tolerate high temperature for a while. After a while, enzymes cannot tolerate these conditions and bacterial growth is decreasing. 900C is so lethal for E.coli cells. Enzymes begin to be degraded at even first 10 min. Bacterial growth cannot be observed at that point.
Control group was used for only comparing.
Oligodynamic action,
Heavy metals can affect bacteria with various ways in even low concentration. Many of them interact with enzyme, protein or amino compounds and then their activity is affected (6). Each kind of bacteria shows different effect for heavy metals. Some genes on their plasmids can have resistance for these. In this experiment, 0,5M Cr3+ , Cr6+ , Mn2+ , Ni2+ , Co2+ , Cu2+ , Fe2+ , Fe3+ , boric acid , Mg2+ and 0,05M Cd2+ were used for observing effects on E.coli cells.
When Table 2 was observed; Cd2+, Co2+, Mn2+, Cr3+, Cr6+, and Fe3+ have high influence onto E.coli cells (order of metals according to effects by diameter relatively). Even so low concentration of Cd2+ and Co2+, they have huge Oligodynamic impact for cells. Especially, 0,05M Cd2+ is so low concentrated solution and owns a strong lethal impact on E.coli bacteria. Also some metals like Fe2+can have weak effects on cells. On the other hand, boric acid and Mg2+ do not seem to have an influence. E.coli bacteria may tolerate these with help of genes in their plasmids.
UV light is a type of light having high energy. UV light can damage cellular structure. Especially damages on DNA can kill bacteria easily. At some microbial applications, a technique as sterilization can be used. At this experiment, Table 3, it can be seen that bacterial growth is not affected by UV rays. Such an observation cannot be expected due to the endospores since this bacterium does not produce spores (7). The main reason can be E.coli bacteria have resistance genes for UV light or the machine producing UV light may not have given light with proper wavelength.
References:
[1] Russell AD. 2003. “Lethal effects of heat on bacterial physiology and structure”.  Science Progress.(86): 115-37. Science Reviews 2000 Ltd
[2] Jay, J.M. (1992). Modern Food Microbiology. 4th Edition. New York: Chapman & Hall. pp. 342–6.
[3] Cowan, Marjorie Kelly (2012). Microbiology: A Systems Approach. 3rd ed. pp. 320–321
[4] David S. Goodsell (2001). “The Molecular Perspective: Ultraviolet Light and Pyrimidine Dimers”. The Oncologist. 6 (3): 298–299.
[5] Fotadar U, Zaveloff P, Terracio L (2005). “Growth of Escherichia coli at elevated temperatures”. Journal of Basic Microbiology. 45 : 403–4.
[6] Harke, Hans-P. (2007), “Disinfectants”, Ullmann’s Encyclopedia of Industrial Chemistry. 7th ed. Wiley. pp. 1–17
[7] (http://www.redorbit.com/reference/escherichia_coli/)

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