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The Affect of Various Lidocaine Concentrations on the Pulsation Rates of Blackworms Lumbriculus variegates
By Michael Diab Abstract: The purpose of the experiment performed was to expose blackworms to different concentrations of lidocaine and observe the pulse rates. The concentrations that the blackworms were exposed to were .1mM, 1mM and 5mM Lidocaine solutions. Lidocaine is a suppressant that deals with heart arrhythmias. Our results from the experiment show that as the concentration of lidocaine increased the pulse rate of the blackworms decreased. This is due to the fact that lidocaine increases the action potential threshold of the pacemaker. Introduction: An organism uses the circulatory system in order to transport and supply nutrients to the tissues and organs of the organism, and to excrete the wastes produced by them. The circulatory system is often stimulated by internal and external stimuli. The internal stimuli vary from hormones to enzymes, to different chemical proteins. The external stimuli could be a physical or chemical stimulus. The two main types of circulatory systems are open and closed. In an open circulatory system, such as that found in most mollusks and in arthropods, there is no distinction between the circulating fluid and the extracellular fluid of the body tissues. This fluid is thus called hemolymph. In a closed circulatory system, the circulating fluid, blood, is always enclosed within blood vessels that transport it away from and back to the heart. Some invertebrates, such as cephalopod mollusks and annelids, and all vertebrates have a closed circulatory system (Raven 2008). Blackworms have a closed circulatory system. Oxygen is obtained through the worms tail and moved down to the head. Chemicals and nutrients can also actively be absorbed from the environment from their skin. The reason that blackworms were chosen for the experiment is because their skin is transparent and blackworms have a large dorsal blood vessel that pumps the oxygenated blood from the tail, to the head of the worm by using muscular contractions that can easily be seen through a microscope (Bohrer 2006). Lidocaine is a common anesthetic and anti-arrhythmia drug that is used throughout the world during surgery. It is used to decrease the sensitivity of electrical impulses in the heart to slow the heart down to a normal rate in humans. Lidocaine can also be actively absorbed through the tail of the blackworms. The goal of the lab was to observe

2 and record the effects that lidocaine has on the pulse rate of blackworms. The question asked was, what effect will lidocaine have on the pulse rate of the blackworms tested. The first alternative hypothesis was that lidocaine would decrease the pulse rate in blackworms. The null hypothesis was that the lidocaine would have absolutely no effect on the pulse rate. The second alternative hypothesis was that lidocaine would increase the pulse rate in blackworms. In an experiment by Y Fujuita, S Endoh, A Sari, and T Yakusawa, Lidocaine suppressed the pulse rate in pigs. (Fujita 1982). And in an experient by W. G. E. Vanteeffelen, Daphne Merkus, Luc J. Bos, Isabelle Vergroesen, and Jos A. E. Spaan, lidocaine increased the pulse rate in dogs (Vanteeffelen 1998). Due to the closed circulatory system that resembles humans, the effects of lidocaine on blackworms should be easily seen. Methods: Control Experiment: The first part of the lab was done to establish a control group for the future experiment. It was also done to examine whether a difference in pulse rates existed between cut blackworms and uncut blackworms. Using a pipette, we extracted 5 -10 uncut worms and placed them into a bowl that was labeled uncut that was filled with approximately with 2 cm of spring water. The same method was used to extract 5-10 cut worms that were placed into a bowl that was labeled cut. We extracted a worm from the uncut bowl and placed it onto a glass slide with a trough. We absorbed excess water with a Kimwipe and waited until the worm remained stable. A cover slide was then placed over the worm. We then placed the slide under a dissecting microscope with the light turned off. Once the worm was located under the lens of the microscope, a student counted the number of pulses that ran through a section of the worm for thirty seconds. To figure out the number of pulsations per minute, the numbers of pulses within the thirty-second span were multiplied by two. We wrote the number into our data table and the pulsations of the worm were counted twice more for 30 seconds each. We did this entire process of counting and observing the worm for thirty seconds again, three times, for two different uncut worms. We recorded the values of each thirty-second interval multiplied by two. Once the three uncut worms were finished being observed, we repeated the entire extraction/observation process for three cutworms. The pulse rates for these worms were recorded on a separate table. The averages of the cut and uncut worms were then calculated as a class and compared. After observing the worms they were placed back into used worm bowls, separated by uncut and cut labels (Bohrer 2006).

3 Lidocaine experiment: For the lidocaine experiment we used three uncut worms and tested the effects of three different concentrations of lidocaine. The concentrations that were utilized were 0.1mM, 1mM, and 2mM lidocaine solutions. The process was much like the control experiment. However, this time prior to measuring the pulse rate, the worms were soaked in their respective lidocaine solutions. For the first set of worms, they were each soaked in 0.1mM of lidocaine solution. They were then washed off with spring water and then placed in the trough of the glass slide. Excess water was absorbed with a Kimwipe and a cover slip was placed on the worm after it had settled. The slide was then put under a dissecting microscope with the light off. Once the student located the worm, they counted the number of pulses for a thirty-second span. Two then multiplied the number observed, giving the average pulsation per minute. The data was recorded and the worm followed the same process twice more. Two other worms endured the same testing and observation. The entire procedure was repeated for the other two concentrations of lidocaine, 1mM and 2mM solutions. The average pulsations were calculated overall for each respective solution. Results: The results of the control experiment data show that there was no difference between the heart rates of the cut blackworms and uncut blackworms. However the lidocaine experiment showed that as the concentration increased, the pulse rate of the worms decreased. Table 1 illustrates that the average pulsation rate for the cut worms was 12.5 bpm, which was lower than that of the uncut blackworms which averaged at 13.2 bpm. The small standard error showed that for cut blackworms it was just 0.63 and for uncut blackworms it was 0.54. Table 1: Average heart rate (bpm) of cut versus uncut worms Type of Blackworm Average STDEV STDERROR

Cut 12.5 2.702871463 0.637072913

Uncut 13.2 2.300191808 0.542160408

Figure 1 illustrates the standard error bars. There is low confidence in figure one because the bars overlap. Thus there is almost no difference between the pulse rates between the cut and uncut blackworms.

Figure 1: The heart rate (bpm) of cut versus uncut worms indicating that there is no difference between the heart rates of cut and uncut worms Table 2 shows that as the concentration of lidocaine increased, the pulse rate of the blackworms decreased. The rates of pulsation were 13.2 bpm, 15.1 bpm, 14.8 bpm, and 9.5 bpm at the respective concentrations of 0mM, 0.1mM, 1mM, and 2mM. The 0mM concentration represented the control pulse of the uncut worms at 13.2 bpm Table 2: Average Pulse rates for uncut worms at respective concentrations of Lidocaine and the Standard errors of each Cut/Uncut worms concentration

Figure two illustrates that as the pulse rate decreased the concentration of lidocaine increased. The standard bars for the 0.1mM and 1mM concentrations of lidocaine overlap, thus showing that there is no significant difference between the concentrations. The

Concentration of Lidocaine 0mM 0.1mM 1mM 2mM

Average Pulsation Rate 13.2 15.1 14.8 9.5

Standard Deviation 2.301 2.143 1.018 1.668

Standard Error 0.542 1.24 0.588 0.963

concentrations of 2mM and the control do not over lap thus suggesting a high confidence and noteworthy difference in the pulse rates of the worms with 0mM, 1mM and 2mM concentrations.

Figure 2: The average pulsation rate for uncut worms exposed to different concentrations of lidocaine. Discussion: After the experiment was done, our results showed that the first alternative hypothesis, that lidocaine would decrease the pulse rate, was supported. This disproved our second alternative hypothesis, that lidocaine would increase the pulse rate, and our null hypothesis, that lidocaine would have absolutely no effect on the pulse rate of the blackworms. Our null hypothesis was disproved due to the fact that not all of our standard error bars in figure 2 did not all over lap. Illustrating that there were noteworthy differences between the control and respective concentrations of lidocaine. Our first alternative hypothesis was supported due to the results of the average pulsations per minute in the different concentrations of lidocaine. The bpm of the worms increased from 13.2 bpm to 15.1 bpm when they were placed in the 0.1mM concentration of lidocaine. Due to the overlapping standard error bars however, that can be neglected because there no statistical difference. Continuing, as the concentration of lidocaine increased from 0.1mM to 1mM to 2mM the pulse rates decreased from 15.1 bpm, to 14.8 bpm, and finally all the way to 9.5 bpm. Since the rates decreased every time, the second alternative hypothesis was disproven. As previously stated the circulatory system reacts to different stimuli. In this case the stimuli was lidocaine. The effects of lidocaine in our experiment showed that as the concentration of lidocaine increased, the pulse rate of the blackworms decreased. Similar experiments have been done that illustrate the same effects in different animals. In an experiment done by Dr. M Mounir and Abou MAdi, lidocaine was shown to suppress arrhythmias and tachycardia from the heart from occurring, slowing and regulating the pulse rate and heart beats in human beings. Lidocaine is a suppressant that causes the sinoatrial node of the heart to have a larger threshold potential to be stimulated, thus slowing the heart rate down (Mounir 1977). Testing different stimuli other then lidocaine can incorporate

6 future studies. Examples could be different type of sugars, drugs, or hormones such as epinephrine. The main sources of error could have been observing the pulses of the blackworms. They were difficult to see and sometimes, movement could have been misunderstood to be a pulse. A way to fix this issue could have been instilling a dye inside the worms to be able to see the pulse rate better. Another source of error couldve been knowing the fact that lidocaine is actually a suppressant. If the chemical we were working with was not known prior to experimenting we may not have been biased in incorporating our data and purposely lowering our data numbers to make them fit. To fix this problem we couldve been revealed the chemical after experimenting was already completed. Literature Cited: Raven, Johnson, Mason, Losos, Singer. The McGraw-Hill Companies Inc. Raven Biology. 9th Edition. United States: McGraw Hill. 1023 p. Bohrer, K.E. 2006. Effects of drugs on pulsation rate of Lumbriculus variegates (blackworms). Pages 127-146, in Tested Studies for Laboratory Teaching, Volume 27 (M.A. ODonnell, Editor). Proceedings of the 27th Workshop/ Conference of the Association for Biology Laboratory Education (ABLE), 383 Pages. Drewes, C. September 2004. Lumbriculus variegatus: A Biology Profile. Iowa State University: C. Drewes; [September 2004]. Available from: Mounir N., Abou-M Adi, Keszler H, Yacoub J. Cardiovascular reactions to laryngoscopy and tracheal intubation following small and large intravenous doses of lidocaine. Pages 12-19, in the Canadian Journal of Anesthesia. Volume 24.,Number 1. (1977)

7 Fujiya Y, Endoh S, Yakusawa T, Sari A:. Lidocaine increases the ventricular fibrillation threshold during bupivacaine-induced cardiotoxicity in pigs. Br J Anaesth 80: 218-222, 1998 VanTeeffelen JW, Merkus D, Bos LJ, Vergroesen I, Spaan JA. Impairment of contraction increases sensitivity of epicardial lymph pressure for left ventricular pressure. Am J Physiol Heart Circ Physiol 274: H187 H192, 1998