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ACKNOWLEDGEMENT The proponents would like to express their heartfelt gratitude and appreciation for the following persons

who helped in the development and fulfilment of this project: To Ms. Maria Fe M. Dumaran, the researchers research adviser, for the extended unlimited assistance extended to the proponents throughout the preparation; To Mrs. Jessica M. Lumapas, Mrs. Gene Pearl A. Luna an Mrs. Kristal G.Entrino for their constructive suggestions and comments for the completion of the study; To Ms. Joan Christella Algallar for her valuable support in the final editing of this research; To Dr.Bernadette J. Nanual, for being the best consultant, for the valuable responses which played a major role in the development of this project, for her ample generosity and for giving her time and effort in entertaining queries, To Marie Rose Campos, for sharing her ideas about the results. To their beloved parents, for their unconditional love, support and being an inspiration whom the proponents dedicate their fruits of hard work; for the undying support and encouragement that they have given, To their fellow Regscian students, for their never-ending support and encouragement, for believing and for the strong friendship that has been founded, To their friends, for lending their helping hands in their own little ways, And lastly to The Big Guy Above, who gives enlightenment, hope and strength which helped the proponents on their way to the success of this project. The proponents are very much grateful for all the things that were imparted to them. Without it, this project would not be successfully accomplished. Ace , Trish and Chin <3

Abstract This study entitled (Gut Content Analysis of the Squid) was conducted to determine the presence of Escherichia coli in the guts of squids found at Magapo, Barangay Central, Mati City. This is deemed to be significant for the health and safety of consumers of marine products at Mati, Davao Oriental. Magapo was chosen as the study area since people living in this place do fishing for their daily living, of which, squids are among the commonly sold products. The experiment was conducted by collecting three squids at different time intervals from a hired fisherman. The gut contents of these squids were sent to the Department of Science and Technology Region XI Office for laboratory analysis. Results showed that the gut contents of squids have Escherichia coli which are above the safety level. These results lead the researchers to conclude that the squids taken from Magapo, Mati City, Davao Oriental is not safe to eat if not cooked on high temperatures. Moreover, it is recommended that similar study be conducted to other marine products such as shells found in the area for further evaluation.

CHAPTER I

INTRODUCTION

The pathogenecity of E. coli: statistics on human health cases caused by E. coli; water-borne and food-borne. E. coli is usually heard in the news and involves contamination of some type. Its bad news and makes people sick. E. coli is a bacterium that lives in the intestines of humans and some other animals. The full name of the bacterium is Escherichia coli, and the average amount you excrete with feces every day averages between 100 billion and 10 trillion. In the sewage treatment industry, the amount of E. coli in water serves as an indicator for how polluted the water isit indicates how much human feces is in the water. However, these bacteria are found only in areas of the body that come into direct contact with the outside world: the mouth, the intestinal tract, etc. If E. coli gets into the wrong areas of the human body, it can cause infections including urinary tract infections, pneumonia, peritonitis, and septicaemia. These diseases can be treated using antibiotics. People are in constant danger of ingesting E. coli from the guts of other animals. They are found anywhere animals are found, so when eating, drinking, or touching something that has been near animals, you take the chance of picking up E. coli. Squids contribute much as our source of food and are commonly sold in the market. It is a popular food resource. People love eating it or who buys squids for their viands would be beneficiary of this study. So that they know that it is safe to eat, most of all, no worries in eating it. Squids eat human feces and their guts probably contain an E. coli.

It is detritivore where they can go near the coast. Where there are many houses and foods. Those houses along the coastline dont have toilets they just made the sea as their toilets. Hence, the tendency that squid can eat the human feces. If so, there is also the probability that E. coli gets into the squid which in turn are eaten by humans. With the necessity of the gut content analysis of the squid, the researcher is prompted to conduct this study.

Background of the Study Upon seeing Magapo, it is observed that the environment is polluted. The wastes of the public market directly flow in the sea. Most of the residents living in Magapo dont have septic tanks and do not observe proper sanitation. Human wastes serve as food to the squid. Human wastes may contain E. coli. The gut of the squid may probably contain an E. coli. Due to this, the researchers were prompted to conduct this study to be able to help the residents and the consumers whether the squid contains E.coli or not.

Statement of the Problem This study was conducted to examine the presence of E. coli in the gut content of squid in Magapo. In this study, the proponents sought to answer the following questions: Is Escherichia coli present in the squid?

Statement of Hypothesis Alternative hypothesis H1: The squid in Magapo, Barangay Central contains E. coli. Null hypothesis H1: The squid in Magapo, Barangay Central does not contain E. coli.

Objectives To determine if there is an E. coli present in the samples.

Significance of the Study

The researchers help the communitys environment indicate whether it is polluted or safe. There is an E. coli present in squids it means that there habitat is polluted and this information is an important input it may serve as an advice to the personnel of health institutions and local officials in planning and implementing appropriate measure to ensure human safety in eating squids in the area. This research may help the people living in Magapo to observe proper sanitation.

The result of this study hopes to give accommodation and helpful insight about the gut content of Squid. will be of help to other researchers who would like to venture into studies of similar nature

Scope and Limitation

This study focused only on the examination of the presence of E. coli in the gut content of squids taken at Magapo, Mati City. Types of E. coli and other areas are excluded. The collected squid samples on the specific area were analyzed by DOST REGION XI to see if there is a presence of E. coli.

Definition of Terms

Gut organisms. Peritonitis the inner

the tube by which bilateral animals transfer food to the digestive

is an inflammation (irritation) of the peritoneum, the thin tissue that lines wall of the abdomen and covers most of the abdominal organs. localized or generalized, and may result infection.

Peritonitis may be

Septicemia - is a serious, life-threatening infection that gets worse very quickly. It can arise lungs, abdomen, time as infections of the: Other tissues. Siphom to transfer liquid from one container to another container to a tube using atmospheric pressure to make it flow. Bacteriostatic - a substance that restricts the growth and activity of bacteria without killing the from infections throughout the body, including infections in the and urinary tract. It may come before or at the same Bone, Central nervous system, Heart and

Sample - The sample units taken per lot for analysis.

Chapter II Review of Related Literature

Squids have soft bodies and usually have two muscular tentacles with eight powerful arms. Those of the giant squids used to catch prey using their 10 metertentacles and arms that reach a length of 3 meters. Has also a rough tongue called a radula, which has teeth on the sides. Squids are also known for having translucent skin and have the ability to change colours in order to protect themselves by blending into the environment or to attract mates during courtships. Its colours come from chromztophores which are pigment cells on the outer layer of the skin. These pigment dots expand and contract to show or hide certain colour like red, yellow, black, green, blue, etc. All kinds of squids are free to swim around in the sea at different depths Giant squids lurk in depths of 600 to 2300 feet under the sea. The squid change in colour to blend with their surrounding in order to survive and protect themselves. The pattern in

which squid moves, pumping water through the mantle, is also how squid breathe when fresh water is pumped in, the squids gills are also refilled with oxygen.

Squids have complex digestive systems. The muscular stomach is found roughly in the midpoint of the visceral mass. From these, the bows move into the caecum for digestion. The caecum,a long, white organ is found next to the ovary or testis. In mature squid, more priority is given to reproduction such that the stomach and caecum often shrivel up during the last life stages. The squids large eyes help it to detect even the smallest amounts of light deep under the sea where it usually dwells. Its diet is composed mainly of fish and crustaceans. The suckers on the squids arm are used to capture and grasp on to prey, it is also known as mandible. Once the food is passed to the mandible by the arms, it undergoes intensive chewing before going into the esophagus. Radula has how of fine teeth that covers a tongue like muscle in the squids mouth. Squids are generally common around the globe, with the exceptions of the Giant squids. For the common species, their numbers are so vast that humans regularly fish for squid. Total body mass of squid far exceeded the mass of humans. It is even predicted that rise of global temperature will speed up metabolism and growth in cephalopods resulting in a squid bloom.

(http://en.wikipedia.org/wiki/Squid)( http://tolweb.org/treehouses/?treehouse_id=4225) E. coli In 1946, Joshua Lederberg and Edward Tatum first described the phenomenon known as bacterial conjugation using Escherichia coli as model bacterium. Escherichia coli are members of a large group of bacterial germs that inhabit the intestinal tract of

human and other warm-blooded animals (mammals, birds). Newborns have a sterile alimentary tract, which within two days becomes colonized with Escherichia coli. More than 700 serotypes of Escherichia coli have been identified. The O and H antigens on their bodies and flagella distinguish the different Escherichia coli serotypes, respectively. The Escherichia coli serotypes that are responsible for the numerous reports of outbreaks traced to the consumption of contaminated foods and beverages are those that produce Shiga toxin (Stx), so called because the toxin is virtually identical to that produced by another bacteria known as Shigella dysenteria type 1 (that also causes bloody diarrhea and hemolytic uremic syndrome (HUS) in emerging countries like Bangladesh) (Griffin & Tause, 1991, p 60,73). Shiga toxin is one of the most potent toxins known to man, so much so that the Centers for Disease Control and Prevention (CDC) lists it as a potential bioterrorist agent (CDC, n.d.). It seems likely that DNA from Shiga toxin-producing Shigella bacteria was transferred by a bacteriophage (a virus that infects bacteria) to otherwise harmless E. coli bacteria, thereby providing them with the genetic material to produce Shiga toxin. The Centers for Disease Control and Prevention (CDC) estimates that every year at least 2000 Americans are hospitalized, and about 60 die as a direct result of E. coli infection and its complications. A recent study estimated the annual cost of E. coli illnesses to be $405 million (in 2003 dollars), which included $370 million for premature deaths, $30 million for medical care, and $5 million for lost productivity (Frenzen, Drake, and Angulo, 2005).

E. coli a food borne pathogen E. coli was first recognized as a food borne pathogen in 1982 during an investigation into an outbreak of hemorrhagic colitis (bloody diarrhea) associated with the consumption of contaminated hamburgers (Riley, et al., 1983). The following year, Shiga toxin (Stx), produced by the then little-known Escherichia coli, was identified as the real culprit. In the ten years following the 1982 outbreak, approximately thirty E. coli were recorded in the United States (Griffin & Tauxe, 1991). As a result, only the most geographically concentrated outbreaks would have garnered enough attention to prompt further investigation (Keene et al., 1991 p. 583). It is important to note that only about 10 percent of infections occur in outbreaks, the rest are sporadic. The CDC has estimated that 85 percent of E. coli infections are food borne in origin (Mead, et al., 1999). In fact, consumption of any food or beverage that becomes contaminated by animal (especially cattle) manure can result in contracting the disease. Foods that have been identified as sources of contamination include ground beef, venison, sausages, dried (non-cooked) salami, unpasteurized milk and cheese, unpasteurized apple juice and cider (Cody, et al., 1999), orange juice, alfalfa and radish sprouts (Breuer, et al., 2001), lettuce, spinach, and water (Friedman, et al., 1999). Pizza and cookie dough have also been identified as sources of E. coli outbreaks.

During E. coli outbreaks, such as the ones in Europe in 2011, WHO has responded by supporting the coordination of information sharing and collaboration through International Health Regulations and the International Food Safety Authorities Network (INFOSAN) worldwide.

Working closely with national health authorities and international partners, providing technical assistance and the latest information on the outbreak. In terms of prevention, WHO has responded with a global strategy to decrease the burden of food borne diseases. WHO developed the five keys to safer food message. The Five keys and associated training materials provide countries with materials that are easy to use, reproduce and adapt to different target

audiences. (http://www.who.int/mediacentre/factsheets/fs125/en/)

In 2006, consumers in the United States experienced a major health scare when bags of raw spinach leaves processed in California were found to contain dangerous levels of a bacteria called Escherichia coli, often abbreviated as E. coli. Hundreds of people were sickened, and several deaths were reported.

The source of the E. coli contamination was eventually traced to specific produce farms located in one county in California. While sales of bagged spinach eventually resumed, the public also became aware of some of the dangers of E. coli contamination and exposure.

It is important to realize that not all E. coli bacteria are harmful to humans. The term E. coli applies to a number of bacteria strains present in animal and human digestive systems. Most E. coli bacteria serve a purpose as digestive aids; they are part of the helpful gut flora responsible for breaking down certain foods into more digestible sugars or proteins.

One particular strain of E. coli, \ is the form of bacteria responsible for the most serious complications associated with contaminated foods and other sources. (http://www.wisegeek.com/why-is-e-coli-so-dangerous.htm).

Most Probable Number Method (MPN) The most probable number (MPN) is particularly useful for low concentrations of organisms (<100/g), especially in milk and water, and for those foods whose particulate matter may interfere accurate Escherichia coli counts. Only viable organisms are enumerated by the MPN determination. The bacteria in the prepared sample in question can be found and are usually attached in chains that are not separated by the preparation and dilution. The following assumptions are necessary to support the MPN method. The sample is prepared in such a way that the bacteria are distributed randomly within it. The bacteria are separate, not clustered together, and they do not repel each other. The growth medium and conditions of incubation have been chosen so that every inoculum that contains even one viable organism will produce detectable growth. The essence of the MPN method is the dilution of a sample to such a degree that inocula will sometimes but not always contain viable organisms. The outcome producing growth at each dilution, will imply an estimate of the original, undiluted concentration of bacteria in the sample. The first accurate estimation of the number of viable bacteria by the MPN method was published by McCrady (1915). Halvorson and Ziegler (1933), Eisenhart and Wilson (1943), and Cochran (1950) published articles on the statistical foundations of the MPN. Woodward (1957) recommended that MPN tables should omit those combinations of positive tubes (high for low concentrations and low for high concentrations) that are so improbable that they raise concern about laboratory error or contamination. De Man (1983) published a confidence interval method that was modified to make the tables below (http://www.unido.org/fileadmin/media/documents/pdf/Agro/MacroLab.pdf)

CHAPTER III METHODOLOGY Locale of the Study

( http://www.mati.gov.ph/maps.aspx) The study was conducted at Magapo, Barangay Central, MatiCity which is approximately 1 km away from the City Hall.

Collection of Squid The researchers collected samples at Magapo, Barangay Central last January 23, 2012. The collected samples were sent to Department of Science and Technology for gut analysis. One parameter is to be measured E. coli count to test if the sample is safe to eat.

Sample Code

Sample Description

Quantity

Container

MIC-2012-01-3897- Squid in PE bag 074 labelled A 2:00 pm 250 g PE bag

MIC-2012-01-3898- Squid in PE bag 075 labelled pm MIC-2012-01-3899- Squid in PE bag 076 labelled pm C: 6:00 250 g PE bag B: 4:00 250 g PE bag

Sampling Technique One shot sampling was used as sampling technique. Care was observed in establishing the sample which is truly a representative of existing conditions, and to handle it in such a way that it doesnt affect its content.

Chapter IV Results and Discussion . In view of the laboratory results of the squid conducted by Regional Standards and Testing Laboratory Davao, the result given in this report was obtained at the time of test and refers only to the particular samples submitted Sample number Mic-2012-01-3897074 Sample Description Squid in PE bag labelled A: 2 pm Parameter Result

Escherichia coli, 23 MPN/G MPN (Conventional Method)

Mic-2012-01-3898075

Squid in PE bag labelled A: 4 pm

Escherichia coli, 38 MPN/G MPN (Conventional Method)

Mic-2012-01-3899076

Squid in PE bag labelled A: 6 pm

Escherichia coli, 23 MPN/G MPN (Conventional)

Sample A was taken at 2pm and it contains 23 MPN per gram. Sample B was taken at 4 pm and it contains 38 MPN per gram. While sample C was taken at 6 pm and it contains 23 MPN per gram, same as Sample A. The study stated that in every sample with their corresponding time shows different MPN or Most Probable Number from labelled A-B-C.

The most probable number (MPN) is particularly useful for low concentrations of organisms (<100/g), standard MPN. This can cause threats to humans. The method used was not intended to be used to isolate and enumerate E. coli serotypes.

E. coli are considered indicator organisms. The presence of indicator organisms in foods processed for safety. When assessing the presence of indicator organisms in a sample, one must assess the results against the tolerance limits specified by government standards or guidelines, health agencies, or a laboratorys or in houses, keeping in mind that established standards and guidelines are specifically linked to the method used to develop these standards. Residence must know about this study. They must observe proper sanitation. Due to their activities, human wastes are eaten by squid containing Escherichia coli, which in turn eaten by human. .

Although it is alarming, but it is good to hear that preparing it with high temperature, specifically 160 degrees Fahrenheit is safe to it. Therefore it means that no matter how the squid is being preserve, E. coli is present.

Chapter V

Summary, Conclusion and Recommendation

Summary and Conclusion

Squids contribute as much as our source of food and are commonly sold in the market. The study aimed to find out if there is a presence of E. Coli in the squid. Most of the residents in Magapo, Barangay Central, Mati City do not observe proper sanitation.

Their wastes are directly release to the sea. All of us cant hide the fact that sea water was already polluted, due to manmade activities. People secretes human feces, there is a tendency that squid can eat human feces. So there is a possibility that E. coli gets into the squid, which in turn eaten by humans. Researchers

One shot sampling was used utilized the presence of Escherichia coli in squid. This sampling was selected for observation over a single period of time. In a Laboratory results, squids (disambiguation) are concluded that has a presence of E. coli.

Recommendation The proponents recommend to the future researcher to replicate the study and make use of other marine resources. Just like shells. The study area must be abundant of your chosen species. The study is open for another research that might discover another substance that squids might contain.

In general, it is important to wash your hands frequently, be careful when handling squids that could be contaminated and cook our food all the way through. This may not protect us from every bacterium out there, but its a good start.

Literature Cited (en.wikipedia.org/wiki/squid) (tolweb.org/treehouses) (Keene et al., 1991 p. 60, 73) (Keene et al., 1991 p. 583) References MedlinePlus: MayoClinic.com: FamilyDoctor.org: MedlinePlus: Hemolytic E. E. E. Uremic Coli Coli Coli Syndrome

MedlinePlus:

Hemolytic

Anemia

http://en.wikipedia.org/wiki/Escherichia_coli Jennifer-Greyhttp://www.professorshouse.com/FoodBeverage/Topics/General/Articles/E--Coli---Just-the-Facts/ http://www.who.int/mediacentre/factsheets/fs125/en/ http://www.wisegeek.com/why-is-e-coli-so-dangerous.htm http://www.google.com.ph/#pq=vinegar+&hl=tl&gs_nf=1&cp=39&gs_id=53&xhr=t&q= can+vinegar+kill+bacteria+called+e.coli&pf=p&sclient=psyab&pbx=1&oq=can+vinegar+kill+bacteria+called+e.coli&aq=f&aqi=&aql=&gs_sm=&g s_upl=&bav=on.2,or.r_gc.r_pw.r_qf.,cf.osb&fp=609cffbfd962025d&biw=1366&bih=65 1

APPENDIX

GUT CONTENT ANALYSIS OF SQUID (DISAMBIGUATION) IN MAGAPO, BARANGAY CENTRAL, MATI CITY

A Research Paper Submitted to Davao Oriental Regional Science High School

In partial fulfillment for the requirements in Research II

Krystel Ann T. Laurente Trishia Dele A. Cabanes Ace G. Francisquete Researchers

Maria Fe M. Dumaran Adviser

February 2012