Antibiotics Lethal Concentrations In An Alternative Animal Model Biology Essay

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Antibiotics lethal concentration in an alternative animal model is such a project where the effect of antibiotics in different concentration is tested on a model organism. In this project antibiotic kanamycin is tested on brine shrimps by looking the number of eggs hatched after adding the antibiotics. This experiment will help to trace the environmental contaminants that have been accumulated in the marine by the usage of chemicals and also by the natural producers like mold and bacteria. . Applications of this antibiotic include the development of drugs in human and veterinary medicine, growth promoters in intensive livestock production, agricultural pesticides, and additives in many consumer and personal care products. After conducting this experiment it was evident that the addition of the antibiotic here Kanamycin will affect the hatching of the brine shrimp eggs negatively. This implies that it will affect the reported to date for aquatic invertebrates and vertebrates are much higher. Several problems limit the usefulness of available ecotoxicity information for assessing ecological risks of antibiotics. Very few species have been assessed for any individual antibiotic; most studies only report concentrations at which extremely large effects occur (usually a 50 % reduction in survival or population growth) in organisms.

Keywords

Kanamycin, brine shrimps, nauplius, negative control, Artemia

Contents

Abstract & keywords …………………………………………...2

Introduction……………………………………………………...4

Objective………………………………………………………....7

Assumption……………………………………………………....7

Materials………………………………………………………....8

Procedure………………………………………………………..8

Observation……………………………………………………..10

Results…………………………………………………………...11

Discussion………………………………………………………..14

Conclusion……………………………………………………….15

Acknowledgement……………………………………………….15

References………………………………………………………..16

Introduction

Antibiotics Lethal Concentrations in an Alternative Animal Model is such a project where we text the effect of different antibiotic concentration in an alternative animal model. For our experiment we took brine shrimps as the animal model.

Brine shrimps are the species of aquatic crustaceans of the genus Artemia which are commonly marketed in the name Sea-Monkeys. Cysts of Brine shrimps are metabolically inactive and can remain in stable condition for years in dry oxygen free conditions, even at below freezing temperature. But in water it will hatch within hours. The biological life cycle of brine shrimps is of one year. The properties like dormant and short life span made brine shrimps to be used in experiments.

Biological Life cycle

Brine shrimps can tolerate varying salinity levels. Their rudimentary nervous /spinal system led researchers to make them as model organism in experiments without concerning animal ethics. (1) .In the natural environment Artemia produces cysts that float on the water surface. These cysts are considered as inactive and do not have any further development as long as they are dry. When immersed on sea water the biconcave - shaped cysts hydrate become spherical and within the shell itself the embryo resumes its metabolism. After 20 hour the outer membrane of the cysts gets burst and embryo appears. The development of the nauplius is completed when the embryo hangs underneath the empty shell and with in a short time period the hatching membrane is ruptured and the free swimming nauplius is born.(2)

Figure 1

Life cycle of brine shrimps

http://www.google.com.sg/imglanding?q=life%20cycle%20of%20brine%20shrimp&imgurl=http://www.fao.org/docrep/field/003/ac062e/AC062E02.gif&imgrefurl=http://www.fao.org/docrep/field/003/ac062e/AC062E03.htm&h=631&w=550&sz=33&tbnid=Ti1FZKGpuWwSjM:&tbnh=137&tbnw=119&prev=/images%3Fq%3Dlife%2Bcycle%2Bof%2Bbrine%2Bshrimp&hl=en&usg=__C-8ogv0gMbL4572NyDZ_15DVyh8=&sa=X&ei=jX0rTO_PGYKYrAfxybTdBw&ved=0CCgQ9QEwBQ&start=0

Toxicology is the study of the adverse effects of chemicals on living organism. Toxicity defines the degree to which a poison causes adverse effect. It depends on dose, duration route of exposure.

In this experiment the chemical that test is antibiotics Kanamycin. Testing the toxicology of Kanamycin and other antibiotics will help to understand whether the antibiotics will affect the marine organism as there are a lot of ways to get antibiotics to be accumulated in the sea water.

The function of a control group in an experiment is to be a constant to check for any effects a given environment has on variables in your experimental group. The control group isolates any uncontrollable phenomena by not being subjected to the unknown variable being tested. In the experiment that had been done a negative control where there is no antibiotic only sea water is used. Since a negative control group is used subjected to conditions a negative result is expected. By using the negative control it will able to compare the experimental group to quantify the effect of an unknown variable to the controls.

Error bars used on graphs are to indicate the error, or uncertainty in the reported measurement. This give an idea of how accurate a measurement is, or conversely, how far from the reported value the true (error free) value might be. Error bars often indicate one standard deviation of uncertainty, but also indicate the standard error.

Objective

To compare lethal concentrations of antibiotics to brine shrimps with those established for rodents for exploring an alternating organisms for preclinical toxicology testing.

Assumption

Brine shrimp are used in the laboratory for testing the toxicity of chemicals. According to the assumption that I made by the addition of antibiotic will inhibit the hatching of the cysts of the brine shrimps. For this I used Kanamycin as the antibiotic. Conducting this experiment we can find the antibiotics that affect the hatching and growth of brine shrimp or other marine organisms.

Materials

Brine shrimp cysts

Talcum tubes

Expender tubes

96 welled microtiter plate

Pipettes

Sea water

Antibiotics (Kanamycin)

Microscope

Weighing machine

Procedure

Prepared the sample of brine shrimp cysts by mixing it with sea water. This sample was pipette into the 96 wells of the microtiter plate in 50µl. Then the number of cysts in the wells is counted.

The solubility of Kanamycin is 50 mg/ml. As the next step weighed 50 mg of antibiotic Kanamycin and dissolved in 1 ml sea water. This is considered as the full strength stock solution. Then performed serial dilution.

For 10 X dilution

Took 10 % of the stock solution that is 1*10/100=0.1ml of stock solution and 90% of seawater that is 1-0.1= 0.9 ml of sea water.

For 100 X dilution

Took 10 % of the stock solution that is 1*10/100=0.1ml of 10 X diluted solution and 90% of seawater that is 1-0.1= 0.9 ml of sea water.

For 500 X dilution

Took 5 % of the stock solution that is 1*5/100=0.05ml of 100 X diluted solution and 95% of seawater that is 1-0. = 0.95 ml of sea water.

For 1000 X dilution

Took 10 % of the 100 X solution that is 1*10/100=0.1ml of 100 X diluted solution and 90% of seawater that is 1-0.1= 0.9 ml of sea water.

In the microtiter plate we are supposed to do two sets of experiment. First and 7th column is considered as the control so added 50µl of sea water. 50 µl of stock solution is pipette to 2nd and 8th column. In the 3rd and 9th column 50 µl of 10 X diluted Kanamycin solution is pipette. 50 µl of 100 X diluted Kanamycin solution is pipette to 4th and 10th column. 50 µl of 500 X diluted Kanamycin solution is pipette to 5th and 11th column. 50 µl of 1000 X diluted Kanamycin solution is pipette to 6th and 12th column.

After adding the antibiotic the microtiter plate was incubated under normal room temperature for one day.

After one day the number of cysts hatched is counted.

Observation

While I am doing the experiment when I pipette the cysts I was able to found that the some of cysts are floating on the sea water. When it hatched some of the brine shrimps move very swiftly some were motionless and some are in developing stage.

Developing stage of brine shrimps

Figure 2

Results

X axis -> concentration of the antibiotic Kanamycin after performing serial dilution

Y axis-> average of brine shrimp cysts hatched

X axis -> concentration of the antibiotic Kanamycin after performing serial dilution

Y axis-> average of brine shrimp cysts hatchedExperiment1

From the two graphs that have been plotted according to the experiment 1 that had been conduct we can derive that when the concentration of antibiotic decreases the hatching of the brine shrimp increases.

Experiment 2

X axis -> concentration of the antibiotic Kanamycin after performing serial dilution

Y axis-> average of brine shrimp cysts hatched

X axis -> concentration of the antibiotic Kanamycin after performing serial dilution

Y axis-> average of brine shrimp cysts hatched

From the two graphs that have been plotted according to the experiment 2 that had been conduct we can derive that when the concentration of antibiotic decreases the hatching of the brine shrimp increases.

Experiment 3

X axis -> concentration of the antibiotic Kanamycin after performing serial dilution

Y axis-> average of brine shrimp cysts hatched

X axis -> concentration of the antibiotic Kanamycin after performing serial dilution

Y axis-> average of brine shrimp cysts hatched

From the two graphs that have been plotted according to the experiment 3 that had been conduct we can derive that when the concentration of antibiotic decreases the hatching of the brine shrimp increases.

Discussion

From the above results it is very obvious that the assumption made matches the results obtained after doing the test. But the average is almost varies with a small values in all the three sets of graphs.

When discussed the graphs obtained at the end of the experiment, the number of eggs hatched in control, where there is no antibiotic is added, is more. Looking to the 1000X diluion where there is less amount of Kanamycin is added, the number of cyst hatched is less than that of control. When comes to 500X dilution of Kanamycin, the number of hatched cysts decreased further from 1000X dilution. Same as that in 100X dilution number of eggs hatched is low compared to 500X dilution, 10 X dilutions the eggs hatched is lower than 100X. When taking into account the stock solution of Kanamycin the number of eggs hatched is very low compared to the rest of dilutions and control. Briefly the number of hatched brine shrimp cysts are in the order of stock< 10 X dilution < 100 X dilution < 500 X dilution < 1000 X dilution < control.

As predicted the presence of Kanamycin will affect the development of the hatched cysts. From the graph it self it is evident that compared to the wells with antibiotic Kanamycin the one with of Kanamycin the hatching of the brine shrimp cyst also decreases. More over the presence of antibiotic affect the mobility of the brine shrimps.

Conclusion

According to the graphs plotted based on the experiment that had done on the brine shrimps it is evident that the number of cysts of the brine shrimps increases as the concentration of antibiotic Kanamycin decreases. This implies that antibiotic Kanamycin will affect the hatching and growth and the mobility of the brine shrimps.

Acknowledgement

I am most grateful to my Supervisor Dr. Alvin Teo who gave us full support in doing this project work. He provided me with inspiration and support in developing student lab investigation. Then next I like to thank my team mates who me full assistance. More over like to be grateful to Republic Polytechnic to let us do this project by giving financial Support.

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