NEW AND IMPROVED RESEARCH PAPER

The Efficiency of Different Growth Stages of Golden Kuhol Pomacea canaliculata Culture Medium for Escherichia coli

A

Partial

Fulfillment to the

Requirement in MT 41 – Research

Rovie T. Valiente

Edith Jane L. Gabo

Caesey Mae A. Yurong

Joymyrn Concepcion R. Gitamondoc

Rhona Lee Asdillo

Janice M. Maglasang

Karan Irma B. McLean

Nathaldane T. Maldo

Silliman University

, 2011

INTRODUCTION

Even before the discovery of microorganisms, diseases were already an issue. And now that these pathogenic agents are known, the issue remains. To somehow control the spread of the threat, the health sector designated specific medical personnel to take specialized action on such. A part of these are medical technologists who play a great role in aiding the physician in the diagnosis of disease. To be able to perform this function, the culture of microorganisms like bacteria is very necessary to obtain knowledge about them.

Background of the Study

            Because of the rising threats of increasing pathogenecity of modern bacterial strains, the need for close vision of these organisms has also increased. Laboratories need to culture bacteria in order for them to identify colonies, the reason being for the illness of a certain person. To artificially allow the multiplication of microorganisms, certain requirements must be met and keen adherence and considerations must be noted.

Besides considering the different environmental factors, another factor to be aware of is the culture medium. Culture media are solutions containing all of the nutrients and physical growth parameters necessary for microbial growth.

As stated in a literature that in making a culture media we consider the required ingredients of culture media such as water, agar, peptone, casein hydrolysate, meat extract, and yeast extract and malt extract to produce an. environment could provide the needed environmental and nutritional setting for them to develop in it. (http: //www.microrao.com/

micronotes/culture_media.pdf)

  One of the commonly used culture medium is agar, which is a solid medium that allows bacteria to grow. This solid medium is useful for isolating bacteria or for determining the characteristics of colonies that would also help in determining treatments for certain diseases. However, the downside in using agar as synthetic culture media is its affordability. This is the root reason why we came up with this research paper; to find ways on how to economize laboratory expenses and a way to it is by looking for a substitute of agar as a culture medium. 
So as we had a further research in finding the means we came about a study that made mention of using beef as a good source of protein in culturing the bacteria: Beef extract is a good source of protein knowing the fact that it is the major component in the composition of muscles, hair, tendons and the physiologic parts of the body; this is the reason why microorganisms efficiently grow in a beef extract. (http: //www.exercise.com/article/nutrition-basics-protein-the-building-blocks-of-lean-muscle).

As we had a through finding more feasible source of protein there was another study who made mention of the possible use of Golden apple snails proteins’ as culture media.As stated in Snails are known t be good sources of proteins because f the known fact that they are composed of 15% protein, 2.4% fat and about 80% water , which also makes them a viable source of food commodity for poor families.(http://housewifeatwork.blogspot.com/2008/01/ginataang-kuhol-or-snails.html)

But even with this possibility it was questioned that snails may not be a good source of such because it is also a vector of known parasites and they have caused problems in the rice fields leading them to be known as the major pests. As cited that, besides the circumstances that they could be possible source of pathogenic diseases, Golden Apple Snails have also been treated as major pests in the rice fields especially in the countries Philippines, Cambodia, Thailand and Vietnam, Due to the harm they have caused the agricultural fields, people have been using different means of eliminating them such as the use f poisons and traps.  (www.getridofthings.com)

In relation to this they have been doing all means to eliminate snails, but the downside to this is that it has shown bad effects on the environmental aspects. This has opened another idea that we could also make an effective way and much environment friendly elimination of such pests. Considering these facts, we could still use snails as means of source of protein, eliminating them at the same time too, knowing that they multiply fast and is very accessible. As stated in a source, the reproducibility of the Golden apple snails is fast and multiplies twice a month, basically by two weeks time and at 25 degrees Celsius they hatch. And in looking at its nutritive values the medium sized snail contains protein, carbohydrates, phosphorus, sodium, potassium, riboflavin, and niacin. (www.bfar.da.gov.ph)

Now that we have found an effective source of protein in making our own culture medium, our next concern is to decide which bacteria we should culture, and looking through the possible specie, we came up with the Escherichia coli. This bacterium is the most common and readily grows in a non-selective media making it much easier to culture.
As stated in a source Escherichia coli which naturally lives in the gut that causes n harm when exposed to the external environment but may cause gastrointestinal diseases such as colitis and diarrhea , is a bacteria that grows in a non-selective medium , in a temperature f 15-45 degrees Celsius. (Medical Microbiology, 2007)This is how we came about our topic in finding less harmful means in eliminating Golden apple snails by using them proteins in making culture media instead of killing them with -chemicals, then culture a non-selective media E. Coli bacteria which could help us in line with our course too as Clinical laboratory scientists.

Statement of the Problem

            Will there be differences in the efficiency of Golden Kuhol Pomacea canaliculata at different stages of growth as a culture medium for Escherichia coli?

Objectives:

1.      To determine which stage of the Golden Apple snail’s life cycle is the most effective as synthetic culture medium for E. coli

2.      To determine if size has to do with the culturing efficiency of the media

3.      Basing on the results of the first objective, the researchers seek to determine whether the protein level in that stage was the highest of the three.

4.      To determine whether differences in protein levels affect E. coli growth

Significance of the Study

The fields of agriculture and medical technology are purposively viewed to be benefited at the end of the study. Golden Kuhol, with its known harmful effects to rice crops, has been the subject to many studies that aim to eradicate it. These methods range from manual (handpicking) to chemical (molluscicides). These proved of no effect because of the aestivation ability of these organisms. During the hot seasons, they try to survive by burrowing, thus, making all these efforts negligible. The prevalence of these organisms caused them to become serious pests in rice-farming. The study aims to provide an alternative way of eradicating them with useful motives.

            It satisfies the medical aspect because of its possibility of replacing the nutrient agar which is of higher cost while retaining its ability to grow bacteria. They also hold an advantage in the fact that they are abundant.

Scope and Limitations

            With the interest of determining which of the developmental stages is the most effective in bacteria culture, the study makes use of the golden apple snails of three different stages- eggs, hatchlings, and adults. For sampling, the researchers consider the use of artificial culture to grow the snails for the purpose of maintaining objectivity and to make sure no external contaminants. 

Using the biuret protein assay it would determine the specific amount of protein. This in turn would be used in interpreting whether there is a correlation between golden apple snail developmental stages to their efficiency in growing Escherichia coli.

            To solely focus on the stages, the research eliminated aestivation as a factor to consider.

Definition of Terms

For the purpose of understanding the terminologies used in the study and to clarify technical terms, the following are defined expressed in measurable procedure:

Conceptual Definition

     Snail eggs: pink aggregations of individual eggs that can be are obtained in rice paddy banks

     Hatchlings: 15-25 days old after incubation

     Adults: sexually mature: 45-59 days old

    Culture Medium:

    Nutrient agar:  

    Agar-agar: an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium

    Biuret method principle:  Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.

   Golden apple Snail (Pomacea canaliculata): are tropical and sub-tropical freshwater snails from the family Ampullariidae ; a mollusk

Operational Definition

    Efficiency: measured by the number of colonies found in the petri dish after the treatment is given

  Treatment: refers to the application of established culture media( nutrient agar, agar-agar) and the produced media/homemade(media with protein extract from eggs, hatchlings, and adult)

  Colonies: refers to the number of bacterial colonies present in ther culture medium and counted through

  Culture medium: a solid medium feed to E. coli in the patri dish

  Developmental stages – stages in the life cycle of the golden apple snails involving the eggs, hatchlings, and the adults

   Snail eggs: pink aggregates of individual eggs of cultured Golden Kohol found in a controlled rice field

   Hatchlings: 17-day old Cultured Golden Kohol from a controlled rice field

    Adults: 50-day old Cultured Golden Kohol from a controlled rice field

   Golden Apple Snail: A mollusk commonly found in the rice field and are considered pests.

 Nutrient agar: a solid medium in culturing bacteria with standardized composition and includes nutrients and minerals

Review of Related Literature

            Bacteria culture has been a tool used by health personnel to diagnose infectious diseases. It provides a reliable way of estimating their numbers or the viable count, allowing the positive or negative identification of the microorganisms. The identification of microorganisms as to the morphology and the antigen has been made possible by the method. When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. Therefore an artificial culture medium must provide all the nutritional components that a microorganism gets in its natural habitat. To be able to meet all these, the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract (http: //www.microrao.com/micronotes/culture_media.pdf)

            Peptones are enzymatic digests of protein used to cultivate non-fastidious microorganisms (http: //www.suboneyo.com/bacteriological-media-ingredients-fermentation-nutrient.html). Another component which is the beef extract complement the nutritive properties of peptone by contributing minerals, phosphates, energy sources and those essential factors missing from peptone (http: //www.bdbiosciences.com/documents/Beef_Extract.pdf). It is prepared from cow`s skeletal muscle & marrow, is highly nutritious & is used in preparing microbiological culture media (http: //www.suboneyo.com/bacteriological-media-ingredients-fermentation-nutrient.html). What is in the beef extract that allows the growth of microorganisms is protein and it forms the building block of most body parts like the muscles, skin, tendons, blood vessels, hair, and cores of bones and teeth (http: //www.exercise.com/article/nutrition-basics-protein-the-building-blocks-of-lean-muscle).

Snails are chemically-composed of 15% protein, 2.4% fat and about 80% water, making them excellent sources of proteins and cheap sources of food for poor families (http://housewifeatwork.blogspot.com/2008/01/ginataang-kuhol-or-snails.html). But parasites like cestodes employ snails as vectors to transmit them from one host to another; thus, eating improperly prepared snails can be detrimental to the body (www.biology.ualberta.ca/parasites/parpub/themes/.../1070103.htm). With this proven mechanism of disease transfer by way of snails, Golden Apple snails have also been viewed as major rice pests in agricultural countries like the Philippines, Cambodia, Thailand, and Vietnam. Methods have been formulated to eliminate them from the rice fields and home gardens. Such include poison, coffee, yeast traps or beer traps, and the removal of the snail`s preferred habitat. Poisons can be made from iron phosphate. These kill them immediately but negative effects can eventually pose harm to the environment. Coffee causes an increase in heart rate and users expose the snails to it over a long period of tie, allowing heart attacks to take place, eventually leading to death. Yeast and beer traps make use of mechanical organisms with their basic requirements such as habitat can also be a method to eliminate them – they lose chances of survival because the environment is unfavourable (www.getridofthings.com). They have become a problem because there were always plenty of them in a certain vegetative area and the number becomes really great that it became hard to eliminate them. They multiply fast and twice a month, the golden Kuhol lays clusters of about 200-500 eggs. After 14 days at 25°C, the first little snails Pomacea canaliculata appear. An average-sized golden Kuhol contains protein, carbohydrates, phosphorus, sodium, potassium, riboflavin, and niacin (www.bfar.da.gov.ph). Optimal temperature is 18-28°C.

            Escherichia coli (what color, in plate, how to obtain and count) is a bacterium present where there is fecal contamination. Non-selective media has been used to grow them and they can live in 15-45° C. They are considered as harmless commensals in the gut. Pathogenecity varies among strains and it has been observed that when outside the body, no harm is inflicted but gastro-intestinal diseases that range from mild self-limiting diarrhea to haemorrhagic colitis (Medical Microbiology, 2007) Growth on culture medium is observed within 10-20 minutes (Cubelo, 2011).

METHODOLOGY

This chapter contains the methods and procedures used in gathering the data needed in this study including the experimental design, techniques, instrumentation and statistical treatment.

Research Design

The study was laid out using Randomized Complete Block Design (RCBD) with three (3) treatments and three (3) replicates. The treatments were as follows:

Treatment 1- Nutrient Agar (control)

Treatment 2 - Kuhol Egg Agar

Treatment 3- Kuhol Hatchling Agar

Treatment 4- Kuhol Adult Agar

Treatment 5- Agar- Agar

Procedures

Standard procedures in media preparation, isolation and incubation of the test microorganism (E. coli) were followed in the conduct of this study.

A. Collection of Escherichia coli

The pure culture of E. coli was obtained from the Philippine National Collection of' Microorganisms, (Biotech Num.1904) National Institute of Molecular Biology and Biotechnology, University of the Philippines at Los Baños College, Laguna. The microorganism was maintained and used entirely in all the treatments of this study.

B. Sterilization of Glassware and Medium

All glass wares to be used in this study are to be pre-sterilized using an All AmericanAutoclave at 15psi for 20 minutes. Medium was autoclaved for 15 minutes only.

C. Preparation of Culture Medium

1. Nutrient Agar

            Thirteen grams (13.0g) of dehydrated Nutrient Broth (Himedia) and 20 grams of shredded gulaman bars were suspended in one liter (1000 ml) distilled water and was boiled completely until it becomes homogeneous prior to sterilization.

Peptic digest of animals tissue ………………….…..... 5.0 g

Yeast…………………………………………………..1.50 g

Beef ………………………………………………….. 1.50 g

Beef extract ………………………………………….. 1.50 g

Sodium chloride ……………………………….……... 5.0 g

Gulaman Bar ………………………………………… 20.0 g

2.Golden Kuhol Agar

Live G. Kuhol were collected from nearby rice field and were soaked overnight in clean water for cleansing before the mollusks were dishelled. Ten grams (10.0g) of the meat was boiled in one liter distilled water. After boiling, the broth was brought to 1 lit.

Kuhol …………………………………………….. 10.0 g

Peptone ……………………………………………10.0 g

Sodium Chloride ……………………..…………… 5.0 g

Yeast …………………………………..………….. 1.5 g

Gulaman Bar …………………………………….. 20.0 g

The materials were boiled until it forms a homogeneous solution prior to sterilization.

3.Agar-Agar

Shredded gulaman bar (20g) was dissolved in one liter distilled water and added with the following:

Sodium chloride…………………………………. 5.0 g

Yeast ......................................................................1.5 g