The aim of this experiment hour was to design a experiment that analyzed the way that some environmental parameters, such as temperature, oligodynamic action and ultraviolet action affect the microorganisms and their rate of lethality. Lethality is the scientific term used to describe the capacity to kill a certain microorganism .
High values of temperature negatively affect the microorganisms, because in those temperatures proteins denaturate as their three dimensional structure is destructed. This leads to the death of the microorganism. Lethal effect of the temperature is associated with two parameters that affect its rate: TDP and TDT. TDP is the thermal death point and is defined as the temperature in which an organism can die in only 10 minutes. TDP is determined by the analysis of a various temperatures and the way they affect the microorganism. TDT is the thermal death time and it is defined as the time amount required for an organism to undergo death in a certain temperature. It is determined by analysing whether a cell is vital or not in specific intervals of time .
Oligodynamic action is the scientific term used to describe the lethality caused in a specific microorganism because of the little concentration of the heavy metals, whose activity’s effectiveness is assisted by the cellular proteins ‘ s high affinity to the metal ions .
There are several ions that affect the microorganisms and their effects in the microorganisms can be negative, neutral or positive. The heavy metals that are dangerous for the microorganisms are Vanadium (V), Arsenic (As), Cadmium (Cd), Anemonite (Sb) and Mercury (Hg). The heavy metals that demonstrate a beneficial effect for the microorganisms are Chromiıum (Cr), Manganese (Mn), Iron (Fe), Cobalt (Co), Nickel (Ni), Zinc (Zn), Molybdenum (Mo), Silver (Ag) and Tungsten (W). The heavy metals whose effect in the microorganisms is neutral are Lead (Pb) and Uranium (U) .
Ultraviolet radiation’s wavelength is included in the range 10-400 nm. It is a successful about causing lethality in vegetative cells, but not as much in endospores. Exposion of the microorganisms to the ultraviolet light makes the microorganisms’s nucleic acids, especially DNA and some particular proteins develop both chemical and physical changes. In the case of DNA, from the 4 base pairs, the thymine undergoes more changes in the presence of UV light. 1 thymine pair in a certain DNA strand fuses when exposed to UV light and leads the formation of a dimer. UV’s effect is limited, because of its low penetrating power and functions only about the microorganisms that are directly exposed to it. UV’s maximum lethal effect is achieved when the optimum time and intensity of exposure are applied in a certain microorganism .
There were three hypothesis set for this experiment. The first one was that E.coli`s thermal death time would be determined in the shortest amount of time where the microorganism`s death occurred and the thermal death point in the lowest temperature where microorganisms`s death occurred. The second hypothesis was that in the presence of a certain element, ig the growth occured it would mean that this element is beneficial for the microorganisms and in the contrary if growth would be inhibited, it means this element is a dangerous one. Additionally in the third experiment, the exposure of the sample toward UV for more than 5 minutes is expected to kill the E.coli.
MATERIALS & METHODS
- Culture of E.coli
- Water bath
- Drigalski spatel
- Bunsen Burner
- Block heater
- Heavy metal solutions
- LB agar plates
- UV source
LETHAL EFFECTS OF TEMPERATURE
Initially the Petri plates were labelled according to the temperature and the period of time the sample of E.coli that is going to be inoculated in the plate is placed in the water bath. Each of the water baths were set up in the temperatures: 50oC, 60oC, 70oC, 80oC, 90oC and 100oC. Our group performed the experiment in the temperature 50OC. After the labelling process has been performed, a control sample is prepared. Firstly 100 μl of E.coli is taken by micropipette and placed inside the Petri dish. After that, Drigalski spatel is sterilized both in alcohol and through the fire of the Bunsen burner. After the spatel cools down, it is used to spread the inoculum through the plate. Then the plate is closed and placed over the bench under the room temperature conditions.The process of spreading plate is repeated 4 times each 10 minutes and for each plate the eppendorf tube containing E.coli is placed in the tube bath in the temperature 50oC and different amount of times, reciprocally: 10 minutes, 20 minutes, 30 minutes and 40 minutes. After the inoculum is over, all the plates including the control plate are incubated in the temperature 37oC for 24 hours.
3 petri plates are prepared by the spread plate method in the presence of the Bunsen burner. Firstly 100 μl of E.coli is taken by micropipette by help of pipetting. Then it is spread by the Drigalski spatel that was firstly sterilized in alcohol and through the Bunsen burner, and cooled down. The plates are positioned upside down till they dry out. Then they are labelled as the control sample or with the name of the metal that will be placed inside. İn the control sample no solution was placed. In one of the plates 20 μl of Zn taken by micropipette is placed inside, but not spreading is performed. On the last plate the same procedure is repeated, but inside it Mn is positioned inside. They are placed in such a way that the agar media absorbs the specific metal. After that all the plates are incubated under the temperature 37oC for 24 hours.
UV LIGHT EFFECT ON MICROBIAL GROWTH
2 Petri plates are prepared by spread plate method in the presence of the Bunsen burner and in each of the LB agar plates, 100 μl of E.coli is placed. One of the plates is labelled as the control one, while the other is labelled as UV sample. The control sample is placed in the room temperature conditions, while the UV sample is placed for 10 minutes under the UV light source, while the other groups performed the same experiment about different amoutn of exposure time, specifically: 10 seconds, 30 seconds, 1 minute, 5 minutes and 20 minutes. After that both of them are placed in the incubator at the temperature of 37oC for 24 hours.
Table1: Determination of TDT and TDP of E.coli
|Control +++ +++ +++ +++ +++ +++|
No growth: –
Table 2: Effect of UV light on the bacterial growth of E.coli.
|UV exposure of E.coli sample||Growth of E.coli compared with the control sample (++++)|
No growth: –
Table 3: Observed growth in the oligodynamic action determination in three different samples of inoculated E.coli
In all the three parts of the experiment the control samples exhibited growth, because their requirments were fulfilled and the temperatures they were inoculated, were appropriate and included in the range of E. coli`s cardinal temperatures. From the first part of the experiment, it can be determined that thermal death temperature is 60OC, while the thermal death point is approximately 10 minutes. From the values represented in the table 1, it is seen that E. coli has exhibited growth in 50OC, although 48OC is the maximum temperature for E.coli. This could have happened, because of an insufficient time of expsoure, and probably if time of exposure was lengthened, the growth would be inhibited. An unexpected behavior occurs also in the case of 100OC temperature, where in the first two periods of 10 minutes, growth has occurred. This can have happened, because of an insufficient amount of exposure or because of the misinterpreting of the growth in the proper zone growth we were studying.The expectations about the experiment related to the sample of E. coli exposed toward the UV of wavelength at 250 nm was that the growth would be inhibited for an exposure time more than 5 minutes, as over 250 nm, this amount of time is able to destroy the molecular bonds that exist in the E. coli `s DNA. The growth could continue during the time exposure of 10 seconds, 30 seconds and 1 minute, because these specific amount of times cannot completely destroy the DNA structure of E. coli and create thymine dimers. In the exposure over 5 minutes of time, DNA structure gets destructed leading therefore to growth inhibition .
According to the scientific literature about the oligodynamic action of zinc in the E. coli sample it is expected that the zinc favors the growth of the sample when present in a low concentration and act as an inhibitory one when present in a high concentration, specifically . In our experiment, the sample`s growth is inhibited in the presence of zinc and the reason why this has occurred is because of the high concentration of zinc related to the total concentration of the E. coli sample .
The way that zinc affects the E. coli is due to its ability to decrease aminopeptidase activity. Zinc also has a positive effect in the microorganisms, such as E. coli, because of its ability to regulate the osmotic pressure, maintain ionic balance and regulate enzymatic activity. When zinc concentration is high it negatively affects the enzymatic activity of those microorganisms, therefore leading to growth inhibition. Manganese is an important element about E. coli and functions as a beneficial factor in our experiment too where the sample has developed growth. Manganese supports the fat and carbohydrate metabolism in the microorganisms and therefore its presence positively supports the growth of E. coli .
- http://www.oxfordscholarship.com/view/10.1093/acprof:oso/9780198515494.001.0001/acprof-9780198515494-chapter-5 retrieved 21.04.2016 from
- http://encyclopedia2.thefreedictionary.com/Oligodynamic+Action retrieved 21.04.2016 from
- http://www.bu.edu/gk12/jan/janpage_files/Lethal%20Effcts%20of%20UV%20Light/The%20Lethal%20Effects%20of%20UV%20Light%20Lesson%20Plan.pdf retrieved 21.04.2016 from
- https://www.researchgate.net/post/How_much_UVlight_exposure_time_is_required_to_disinfect_a_laminar_flow_hood retrieved 21.04.2016 from
- https://www.researchgate.net/publication/226424873_The_effect_of_zincII_on_the_growth_of_E_coli_studied_by_microcalorimetry retrieved 25.04.2016 from
- https://www.terrapub.co.jp/onlineproceedings/ec/03/pdf/BR_03057.pdf retrieved 21.04.2016 from