Journal Rtw- Group 3

Isolating Helminth Eggs using Gasoline as a Substitute to Ether in the Formalin-Ether Concentration Technique Erylle P. Cadiente Rhubilyn L. Landero Rhea F. Suberon Maria Rheena Flor C. Zamora Bachelor in Medical Laboratory Science, University of Immaculate Conception, Father Selga St., Davao City

Abstract The Formalin- Ether Concentration Technique is a very useful and effective procedure in the diagnosing parasitosis of man but is seldomly used in the clinical laboratory since ether can only be obtained in small due to existing Laws regulating its production and distribution because it is expensive, flammable, volatile, produces anesthetic vapor and explosive (Robert et al., 1996). Gasoline (Regular, Special, Unleaded) were used and proven viable for the substation of Ether in the said Concentration Technique introducing the FormalinGasoline Concentration Technique. 200 clinical known positive fecal specimens were used in this comparative descriptive, experimental type of study wherein four solvents were being compared in the natural setting of the procedure. Results as treated with F-test, ANOVA, Duncan’s and Scheffe’s in the statistics show that there is no significant difference in the effectiveness in the parasitic recovery rate and macroscopic clarity rate of the sample preparation between ether and the three types of gasoline. There is a significant difference in terms of microscopic clarity rate for Gasoline- Regular compared the effectiveness of other solvents. Thus, Gasoline- Special and Gasoline- Unleaded viable in substituting ether in the concentration technique considering the parameters in assessing their effectiveness. Key Words: FECT, FGCT, Ether, Gasoline, Concentration Technique

Introduction In isolating parasites that can be found in the fecal material, several procedures may be used in order to diagnose the presence of a certain underlying pathologic conditions due to parasitosis. The procedures to be used may range from the simple Direct Fecal Smear (DFS) to the more complex procedures of fecal concentration techniques. The simplicity or the complexity of the procedure used will greatly affect the amount of parasites recovered (Zeibig, 1997). Gasoline is a promising substitute for ether since gasoline is of the same type of compound as that of ether. Both gasoline and ether are organic hydrocarbon compounds, meaning they are made up of Carbon, Hydrogen, and Oxygen atoms. Furthermore, they are both of almost the same Density or Specific Gravity, which is very important in the separation of the Fecal Plug formed in concentration techniques. Gasoline is also far more available than Ether, thus making it a very viable substitute. In the Philippine setting, FECT is one of the most effective procedure used to diagnose different types of intestinal parasitosis. It is a sedimentation method to separate parasitic elements from fecal debris through centrifugation (Robert and Jonovy, 1996). Although FECT is the best procedure in isolating parasites present in the feces, since this technique provides less or very minimal alterations to the organisms present in the stool sample and increased recovery of Helminth eggs as well as protozoan cysts, it is seldom performed because of the unavailability of ether. Ether can only be obtained in small amounts since there are Laws that regulate the production, distribution, and trade of ether, because it is extremely flammable, is highly volatile, produces anesthetic vapor, and forms explosive peroxides when exposed to light (Robert and Jonovy, 1996; Boswell and Collins, 1996). Medical Technologists working in the Parasitology Section of the Clinical Laboratory opt to find substances to substitute ether or use the less effective method, Direct Fecal Smear, to overcome these disadvantages, since pursuing the performance of FECT in diagnosing parasitosis implies an additional cost to the laboratory, as well as rendering it unavailable to everyone (Robert and Jonovy, 1996). In this research paper, the study routinely used Formalin-Ether Concentration Technique procedure and the Formalin Gasoline Concentration Technique for isolating parasites from known samples, and compare which among the three types of gasoline (Regular, Special, Unleaded) will yield the best result in terms of parasitic recovery rate and sample clarity rates (macroscopic and microscopic) were studied.

Materials and Method This research is a comparative descriptive, because there were four kinds of solvents whose characteristics are being compared in the natural setting of the procedure. Experimental that it examines the effect of substituting different types of gasoline to ether in the FECT. This research is a non-probability type of study. The subjects of this research were the respondents or the ten individuals from which the positive samples came from. Ten individuals with known cases of parasitosis were selected from the selected families of Brgy. 5A- Bankerohan, Davao City used in the analysis of the viability of the gasoline types as substitutes to ether. Collection of specimen was done from ten individuals in Barangay 5-A, Bankerohan, Davao City with known positive fecal samples. The experimental procedures involved in the study were conducted at the 4th floor Microbiology Laboratory, University of the Immaculate Conception, Fr. Selga st., Davao City, which provided a highly controlled laboratory setting performing the Formalin-Ether Concentration Technique and Formalin-Gasoline Concentration Technique. The laboratory was provided with all the materials needed in the study except the three types of gasoline which were provided by the proponents.

Results and Discussion Table 1.1 Summary of Mean for Recovery Rate Sample 1 2 3 4 5 6 7 8 9 10 Mean

ETHER REGULAR SPECIAL UNLEADED (%) (%) (%) (%) 100 100 100 100 100 80 20 20 100 100 100 100 80 80 100 100 100 100 80 80 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 98 96 90 90

Table 1.1 shows the summary of the means of the recovery rate for each type of solvent used in each sample. In each sample, there are five repetitions of each solvent used, and the means of the trials are plotted under their corresponding solvent. After which, a mean is derived from the means of the trials in each sample under each solvent. These final means are those that are found plotted under table 1.2 and are tested using the F-test for computed F-ratio and probability F-ratio under the significance level of 0.05 in order to decide whether to accept or reject the null hypothesis or if there is a significant difference or not. Table 1.2 F-Test for the Significance of the Difference Between the Four Formalin-Concentration Techniques when Analyzed in Terms of Recovery Rate Variables

Mean

Ether

98

Regular

96

Computed ratio

Accept Ho 0.486

Special

90

Unleaded

90

F- Probability F- Decision on Ho ratio @ 0.05

0.694 Not Significant

Table 1.2 presents the test for the significance of the difference between the Four Formalin-Concentration Technique when analyzed in terms of recovery rate. From the table, the mean for ether is 98 %, regular gasoline is 96 %, special 90 % and unleaded is 90 %. Since there are four variables to be compared, one way Analysis of Variance was used. Further analysis would show that the computed F-ratio yielded a value of 0.486 with a probability F-ratio of 0.694. For the computed F-ratio to be significant, the probability F-ratio must be less than or equal to 0.05 level of significance. In this case, the probability F-ratio is greater than 0.05 level of significance, therefore, there is enough evidence to accept the null hypothesis of no significant difference. This means that there is no significant difference between the recovery rates of the four Formalin Concentration Techniques. This implies that the four concentration techniques have the same effect in

terms of recovering parasite eggs from fecal samples. Although there is a difference between the values of the four means that are being compared, these values are not statistically significant.

Table 2.1 Summary of Mean for Clarity Rate (Macroscopic) ETHER REGULAR SPECIAL UNLEADED Sample (%) (%) (%) (%) 1 86.67 86.67 80 66.67 2 100 33.33 33.33 53.33 3 66.67 33.33 66.67 33.33 4 93.33 66.67 66.67 100 5 80.00 80.00 100 66.67 6 66.67 66.67 33.33 33.33 7 100 100 73.34 93.33 8 86.67 93.33 93.33 100 9 93.33 93.33 73.34 93.33 10 93.33 86.67 93.33 80.00 Mean 86.67 74.00 71.33 72.00 Table 2.1 presents the summary of the means of the macroscopic clarity rate for each type of solvent used in each sample. In each sample, there are five repetitions for each solvent used, and the means of the trials are outlined under their corresponding solvent. After which, a final mean is derived from the means of the trials in each sample under each solvent. These final means are those that are found plotted under table 2.2 and are tested using the F-test for computed F-ratio and probability F-ratio under the significance level of 0.05 in order to resolve whether to accept or reject the null hypothesis or if there is a significant difference or not. Table 2.2 F-Test for the Significance of the Difference between the Four Formalin-Concentration Techniques when analyzed according to Clarity (Macroscopic) Variables

Mean

Computed ratio

F- Probability F- Decision on Ho ratio @ 0.05

Ether

86.67

Regular

74.00

Accept Ho 1.081

Special

71.33

Unleaded

72.00

0.370 Not Significant

Presented on table 2.2 is the test for the significance of the difference between the four formalin concentration techniques when analyzed according to clarity (macroscopic). The table shows that ether has the highest mean of 86.67 followed by regular gasoline 74.00, unleaded gasoline with 72.00 and special gasoline with 71.33. To test the significant difference between the four variables that are being compared, one way analysis of variance was utilized. The table revealed that the computed F-ratio is 1.081 and the probability F-ratio is 0.0370 at 0.05 level of significance. Since the probability F-ratio is greater than 0.05 level of significance, the null hypothesis of no significant difference is accepted. This means that there is no significant difference between the means of the four Formalin Concentration Techniques in terms of macroscopic clarity rates. This implies that the four formalin concentration techniques namely: formalin-ether, formalin-regular, formalin-special and formalin-unleaded have similar effects in terms of macroscopic clarity rates. The lack of significant difference between the four variables strongly allude that the three types of gasoline are viable for substitution to ether in the Formalin-Ether Concentration Technique in terms of the macroscopic clarity of sample preparation. Table 3.1 Summary of Mean for Clarity Rate (Microscopic) ETHER REGULAR SPECIAL UNLEADED Sample (%) (%) (%) (%) 1 32 28 48 44 2 40 36 40 40 3 68 28 48 56 4 44 20 20 40 5 24 24 40 36 6 60 60 60 64 7 20 20 20 52 8 52 24 60 64 9 56 20 56 52 10 20 20 32 40 Mean 41.60 28.00 42.40 48.80

Table 3.1 exhibits the summary of the means of the microscopic clarity rate for each type of solvent used in each sample. In each sample, there are five repetitions of each solvent used, and the means of the trials are plotted under their corresponding solvent. After which, a mean is obtained from the means of the trials in each sample under each solvent. These final means are those that are found mapped under table 3.2 and are tested using the F-test for computed F-ratio and probability F-ratio under the significance level of 0.05 in order to decided whether to accept or reject the null hypothesis or if there is a significant difference or not.

Table 3.2 F-Test for the Significance of the Difference Between the Four Formalin-Concentration Techniques when Analyzed According to Microscopic Clarity Variables

Mean

Ether

41.60

Regular

28.00

Computed ratio

Reject Ho 3.928

Special

42.40

Unleaded

48.80

F- Probability F- Decision on Ho ratio @ 0.05

0.016 Significant

Table 3.2 displays the test for the significance of the differences between the four formalin concentration techniques when analyzed according to microscopic clarity. From the table, unleaded gasoline has the highest mean among the four concentration techniques with a value of 48.80, second in line is special gasoline with a value of 42.40, followed by ether with a mean of 41.60, and the lastly, regular gasoline with 28.00. The table shows that the computed F-ratio is 3.928 and the probability F-ration is 0.016 at 0.05 level of significance. Since the probability F-ratio is less than 0.05 level of significance, then the null hypothesis of no significant difference is rejected. This means that there is a significant difference between the Microscopic Clarity Rates of the four Formalin Concentration Techniques. To determine which of the four techniques differ significantly, Scheffe multiple comparison was applied. This technique revealed that there

is a significant difference between regular gasoline and ether in favor of ether, there is also a significant difference between regular and special in favor of special, and there is a significant difference between unleaded and regular in favor of unleaded. However, there was no significant difference between the three concentration techniques namely; formalin-ether, formalin-special and formalin-unleaded. This result particularly entails that the two types of gasoline, Special and Unleaded are viable substitutes to Ether in the Formalin-Ether Concentration Technique. With regards to the Regular Gasoline, since among the four solvents it has the lowest mean value, it is implied that it is not viable as a substitute to Ether in the Formalin-Ether Concentration Technique in terms of microscopic clarity of the sample preparation.

Conclusion Based on the results presented, there is no significant difference in effectiveness in terms of parasitic recovery rate macroscopic clarity rate of the sample preparation between ether and the three types of gasoline, namely: Regular, Special and Unleaded when used as solvents in the FECT and FGCT in isolating helminth eggs. There is a significant difference in effectiveness in terms of microscopic clarity rate between Regular-Gasoline as compared to Ether, Gasoline-Special and Gasoline- Unleaded when used as solvents in the FECT and FGCT in isolating helminth eggs.

Recommendations In this study, the researchers would like to recommend the following: 1. Recommending the use of Gasoline-Special and Gasoline-Unleaded as a substitute to ether in Formalin- Ether Concentration technique. 2. It is recommended to conduct research and experimentation to test the protozoanisolating capacity of gasoline. 3. Recommendation of the viability of the study to concerning health and/or academic agencies for support and promotion of usage of Gasoline-Special and Gasoline- Unleaded as substitute to Ether in FECT in the clinical laboratories. 4. The researchers would want to recommend that multiple related tests be performed in the future to completely evaluate the helminth-isolating capacity of gasoline.

Acknowledgement Sincere gratitude to all the people who greatly contributed in the success of this research paper mostly to the Almighty God and to the parents of the authors. To Adorico M. Aya-Ay, PhD, Avee Joy B. Dayaganon, RMT, MSMT, John Mark De Real, RMT, Hon. Edgardo Ibuyan, Jr., S. Ma. Carolina Perales, RVM, Maritess Saco, RMT, Micheline Phoebe Pangilinan, M.D., CFP, Ursula Quiban, PhD, Carlito Yurango, Venchie Badong, MAT, Iven Christ Castromayor, RMT, Desiree Victorino, RMT, and Ace Ronald Sarabia, RMT.

References Robert, Peter J. and Jonovy, Helen M. (1996). Basic Parasitology. Manila, Philippines; Pearson Education Asia Markell, John S. (2002). Chemistry for Health Sciences. Manila, Philippines; Pearson Education Asia. Belizario, Vicente Y., M.D., MTM&H and de Leon, W.U., RMT, DAP&E, MPH.(2004).Philippine Textbook of Medical Parasitology (2nd Edition).Manila,Philippines; The Publications Program.