"GOLDEN KUHOL" (Pomacea canaliculata) AS A SUBSTITUTE PROTEINSOURCE IN THE CULTURE of Escherichia coli

"GOLDEN KUHOL" (Pomacea canaliculata)

AS A SUBSTITUTE PROTEINSOURCE IN THE CULTURE of  Escherichia coli

General Objective:

This study primarily aimed to utilize "Golden Kuhol" as an alternative protein source to create a non-synthetic medium for bacterial growth. Specifically this study aimed to answer the following questions.

1. What is the effect of the substrate using "Golden Kuhol" as protein source in growing E. coli bacteria as shown by the number of colony growth?

2. Is there any significant difference between the effects of commercial nutrient Agar and "Golden Kuhol” medium as source of protein in bacterial growth?

Null Hypothesis

There is no significant difference between the effects of commercial nutrient Agar and "Golden Kuhol” medium as source of protein in bacterial growth.

Scope and Delimitation

This study was conducted purposely to determine if "Golden Kuhol" can be usedas an alternative protein source in a nutrient agar.

Research Design

This study was laid out in a Randomized Complete Block Design (RCBD) withthree (3) treatments replicated six (6) times. The data gathered were interpreted bymeans of Analysis of Variance (ANOVA).

Findings

Based from the gathered data, the following findings were revealed:

1. The results showed that treatment 1 (control) attained the highest number of colonies with an average of 333 counts after 48 hours of incubation. Treatment 2 (Kuhol& Agar) attained an average number of 295 colonies considered not significantly different from the control treatment. Treatment 3 (pure Agar) attained no growth at all.

2. Statistical analysis using ANOVA revealed that the computed F-value of 4.4 is less than the tabular F-value of 7.56 at 1% level of significance, thus null hypothesis is accepted. This further revealed that there is no significant difference between the effectsof commercial protein Agar and "Golden Kuhol" medium as source of protein in bacterial growth.

Conclusions:

Based on the findings of this study, the researchers concluded the following:1."Golden Kuhol"(Pomacea canaliculata) is an effective alternative protein source to grow  Escherichia coli as shown by the number of colony growth.2.There is no significant difference between the effects of commercial protein Agar and "Golden Kuhol" medium as source of protein in bacterial growth.

Recommendations:

In view of the above findings and conclusions, the following recommendations were formulated:1.It is recommended that further research be conducted for other possible protein sources for the protection of bacterial culture medium.2.Other researches can also be undertaken to find other ways of utilizing kuhol.3.It is also recommended that other researches be conducted for other substitutes to beef as a source of protein in culturing bacteria.

Chapter I THE PROBLEM AND ITS SETTING Introduction

A fundamental method of studying bacteria is by culturing in liquid media or on the surface of media that have been solidified by agar. Media contains nutrients, varying from simple sugar to complex substances such as meat growth. Most media, used for isolation, growth and cultivation of microorganisms is solid media. This media contain agar – a gel forming polysaccharide mixture from several species of red seaweed {agarophytes}. {Quimio and Quimio, 2001}Most culture media are formulated to identify bacterial species of interest. For several purposes, the most commonly used media for bacterial isolation and growth is nutrient agar. The main component of a nutrient agar is beef extract and peptone.{Fernandez, et. al.}There are several synthetic and natural media which are being used in the cultivation of fungal pathogens. A synthetic medium is one which the exact chemical composition of the ingredients is known. In non-synthetic medium, the precise composition of some or all nutritive substances used, are not definitely known. { Quimioand Quimio 2001}. There are commercial dehydrated media sold in the market, but cheaper media can be prepared easily from available natural products. Dehydrated media are in powdered form, ready to be prepared by merely restoring the water with distilled water and sterilizing it. To use media from locally available sources is a challenge to most individuals and professionals working with different microorganisms purposely to lower the cost of the

 procedure, without hampering the quality of results obtained. The Golden Apple Snail { Pomacea canaliculata} also known as “Golden Kuhol”,is a large freshwater snail native to tropical and subtropical South America. Since the1980’s, this snail has become a serious rice pest in Asian countries because it damages the young rice seedlings. {Yusa and Wada 1999}. As a known source of protein, the possibility of this pest as substitute for beef extract in nutrient agar is being look into.2

STATEMENT OF THE PROBLEM

This study primarily aimed to utilize "Golden Kuhol" as an alternative protein source to create non-synthetic medium for bacterial growth. Specifically this study aimed to answer the following questions: I What is the effect of the substrate using "Golden Kuhol" as protein source in growing Escherichia coli as shown by the number of colony growth?2. Is there any significant difference between the effects of commercial nutrient agar and "Golden Kuhol" medium as source of protein in bacterial growth?

Null Hypothesis

There is no significant difference between the effects of commercial nutrient agar and "Golden Kuhol" medium as source of protein in bacterial growth.

Significance of the Study

Findings of this study are beneficial to the readers for they will be able to know significantly the use of "Golden Kuhol" in the growth of bacteria. This study is significant to the following:

Farmers

. This study will offer significant use of Gold Kuhol while at the same time help in eradicating them as rice pest.

Food Processing Companies

. Findings in this study will give the food processing companies an idea on culturing bacteria at a cheap cost. Food processing companies MC cultured bacteria in the production of different types of food such as yogurt and cheese

Microbiologist

. This study will provide insight and basis for studying other pest, as an alternative media component

Teacher

. Findings of this study will be an additional knowledge to the teachers, which they could share to their students.

DEFINITION OF TERMS

For the purpose of clarification, the following terms were defined.

Colonies

. It refers to the number of bacterial colony present in the culture medium.

Culture medium

. A solution feed to all microorganisms. 

Treatment

. It refers to the application of the different amount of media used.

Golden Kuhol.

A mollusk, considered as one of the major pests in the rice fields in the Philippines. It is scientifically known as Pomacea canaliculata.

Nutrient Agar

. It refers to a solid medium in culturing bacteria.

Agar-Agar.

A gel from seaweed extract, widely used for growing microorganisms in laboratories.

Substrate

. A substance that is acted upon especially by an enzyme, in a biochemical reaction.

Scope and Delimitation

This study was conducted purposely to determine if "Golden Kuhol" (Pomaceacanaliculata) can he utilized as an alternative protein source

The numbers of colonies among treatments were counted after 48 hours of incubation for ruling the affectivity of the golden kuhol as an alternative protein source. The experiment was conducted within the period of six weeks from January to March 2005 at the Camarines Norte State College main campus laboratory

Endnotes

Fernandez. W.L. 0.41., "General Microbiology". 1995. 2' edition. Institute of Biological Science, UPLB.Quimio. A.J. and T.II. Quimio. 2001. "Introduction to to Fungal Pathogens".University of the Philippines, Los Banos Laguna. College of Agriculture PublicationOffice.Encarta Dictionary Tools. 2003. Microsoft Word

Chapter 2REVIEW OF RELATED LITERATURE AND STUDIES

The related literature and studies gathered by the researchers enable them to obtain wide range of knowledge, which are valuable guide in conducting this studySignificant data derived from different sources were utilized and adopted for theimprovement and success of this study.

Related literature

The following literatures were found by the researchers to be highly significant to the present study. The golden snail

(Pomacea canaliculata) was introduced from Florida and Latin American to Taiwan in the early 1980's to start escargot industry. The snail fast breeding and adaptability, as well as its high protein content, made it an ideal dietary supplement. When initially introduced to the market, the golden kuhol was expensive. As the market disappeared, snails were already released into the wild. These snails soon spread to rice fields, entering through waterways and irrigation canals. The golden kuhol has a bronchial respiration system that allows it to breathe under well as a lung that respirates air. This allows the snails to adapt quickly to changing conditions and expands its ability to search for food, prompting the snail to leave the water when the food supply below the surface is inadequate. Female snails lay egg up to 500 eggs per week, causing population explosion. Such adaptations, along with high population densities, have made snail a serious pest in many areas of cultivated rice land in Asia

(Case Study of Golden Snail, U.S.

Department of State).

Professor Moffert (1999) found that golden snails are well edible and are often considered a protein rich-delicacy. The nutritional value of golden snails is relatively high. More precisely, the protein content of snail can make them a good protein source for humans. Growth medium also called culture medium, or nutrient broth, is a solution feed to all microorganisms after sterilization autoclaving 15psi for 15 minutes; others contain exact quantities of known inorganic salts and one or more organic compounds (synthetic chemically defined media).According to Anzaldo (1987), agar is one of the indispensable extract obtained from genus Gracilaria belonging to red seaweeds. The value of agar lies in its stronglyhydrophilic colloid and high gel strength properties. It is used as a suspending,stabilizing, thickening or gelling agent if desired. Its most important service to making, a bacteriological culture medium.

Related Studies

The researchers have found the following studies to be relevant and significant to the present study: Hernandez, et.al (2002) conducted a study about "The Different Concentration of Ginger Extract in Escherichia coli”. Their study focused on the culturing of bacteria by the use of agar as a solid medium and meat extract as nutrient broth. Bacteria were cultured and produced in 18 culture media. The experiment resulted to an oval and round shape bacterial colony. As to colony size and number, the treatment I (ti) obtained the highest count mean of 95 colony count. In the study conducted by Adrian Farolan, et.al (2001) entitled "Pulverized Golden Kuhol as Protein Source in Culturing Bacteria" they used different percent of  pulverized kuhol in media preparation. They concluded that the treatment having highest percentage of pulverized kuhol obtained the highest number of bacterial colonyTherefore, pulverized golden kuhol can he a possible source of protein in culturing bacteria.

Synthesis of the State-of-the-Art

The different studies cited were found to be significant to the present study due to their similarities in the conduct of this research. In the study of Hernandez, et.al. they cultured bacteria using agar as solid medium and beef extract as nutrient source, while the present study used golden kuhol broth as nutrient source. Previous study by authors used concentration of ginger as anti-microbial while present study focused only in bacterial growth. Both present and previous study used Es cherichia coli as test micro-organism. In the study of Farola, et.al. they used pulverized "golden kuhol" while the present study used of "golden kuhol broth" in culturing bacteria. Both studies focused on the utilization of "golden kuhol” using experimental research as method.

Endnotes

Case Study of Golden Apple Snail, I.T.S. Department of State. 2003. pp.14-16.Rice Technology Bulletin. 2001. No.33Hernandez, et.al. "Different Concentration of Ginger Extract In Escherichia coli",Unpublished Undergraduate Thesis, CNSC, Dact, Camarines None.Adrian Farolan, et.al., “Pulverized Golden Kuhol as Protein Source in CulturingBacteria", Unpublished Undergraduate Thesis, Ateneo de Naga. 2000

Chapter 3METHODOLOGY

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

Research Design

The study was laid out using Randomized Complete Block Design (RCBD) withthree (3) treatments and six (6) replicates.The treatments were as follows: Treatment I- Nutrient Agar (control) Treatment 2 - Kuhol Agar Treatment 3 - Agar-Agar only

Materials and Instruments

The materials and instruments used were as follows:

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 andBiotechnology, University of the Philippines at Los Hallos, College Laguna. Themicroorganism was maintained and used entirely in all the treatments of this study.

B. Sterilization of Glassware and Medium

All glassware used in this study were pre-sterilized using an All American Autoclaves at 15psi for 20 minutes. Medium was autoclaved for 15 minutes only.

C. Preparation of Culture Medium1. 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 …………………….....5.0g

Yeast…………………………………………………..1.50 g

Beef ………………………………………………….. 1.50 g

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

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

Gulaman Bar ………………………………………… 20.0 g

2.

G. Kuhol Agar

Live G. Kuhol werecollectedfromnearbyricefield and weresoaked overnight inclean water for cleansing before the mollusks were dishelled. Ten grams (10.0g) of themeat 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 tosterilization.

3.Agar-Agar

Shredded gulaman bar (20g) was dissolved in one liter distilled water and addedwiththefollowing:

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

Peptone …………………………………………….. 5.0 gThe mixture was boiled thoroughly until it liquefied prior to sterilization

D. Sterilization

All media were sterilized using an autoclave (All American) at 15-psi for 15minutes. Most media used in micro-biological and pathological work are sterilized at this temperature/time schedule.

E. Maintaining Pure Culture

1. To maintain bacteria, it was transferred periodically to fresh agar slope or stab and kept at 40 c.. Agar slope culture consists of organisms grown in test tubes or small bottles containing agar which has been allowed to solidity as slant. Stab culture consists of organisms grown in as agar medium inoculated by plunging a straight wire vertically into the center of the tube.

2. Aseptic techniques were followed while making the transfer.3.Two tubes were held by the left hand, palm up, with the culture tube (tube no. 1) between the index and middle finger and the new agar slope (tube no. 2) between the middle and the fourth fingers. The tubes were secured with a thumb.4.Wire loop held by the right hand was sterilized by passing the loop directly through the fire from an alcohol lamp.5. Plug of the inner tube (tube no. 2) was removed with the little finger and the palm of the right hand and the plug of the outer tube (tube no. 1) with the middle and fourth fingers.

6. The test tube mouth was flamed each time before replacing the cotton plug. During the process, the sterilized wire loop and the inner portion of the plug was not allowed to come in contact with any unsterile object.7.The wire loop was inserted in culture tube no.1 to scrape very little amount of the bacterial growth. This then, was streaked lightly on the surface of the agar slope of tube no. 2 starting from the bottom to the top. The mouths of the tube wereflamed.8.Plug no. 1 was returned properly to tube no. 1 and so as to tube no. 2.9.The wire loop was again sterilized directly to the wire and splattering wasavoided.

10. The pure culture was incubated at room temperature for 48 hours.

F.Serial Dilution

In a serial dilution, the original inoculums is diluted in a series of dilution tubes, A10m1 distilled water pour into original inoculums bacteria, and its succeeding dilution tubewill only have 9m 1 of distilled water. One (1.0m1) of inoculums sample was transferred to second tube with 9m1 distilled water by using sterilize pipettes.

G. Pour Plate Method

a. Inoculate empty plate with 1mL from the serial dilution. b. Add melted agar medium using aseptic techniquec. Swirl to mix.d.Allow the bacteria to grow for 48 hours.

H. Observation of Bacteria

Bacterial growth observation will be done once by counting the numbers of  bacterial colonies growth in each petri dishes after 48 hours of incubating

COLLECTION OF PURE CULTURE(E. coli)STERILIZATION OF GLASSWARESSTERILIZATION AND DISINFECTIONOF CULTURE MEDIUMINOCULATION OF NUTRIENTAGAR MEDIUMSERIAL DILUTION(10

-5

)POUR PLACE METHODINCUBATION FOR 48 HOURSEVALUATION(COLONY COUNTING)Figure 7

Flow Chart

Statistical Treatment

The data gathered were interpreted by means of One-Way analysis of VarianceF-ratio was used to determine the significant difference between the treatments.The formula used was:F=MSTMSEWhere:MST refers to mean sum of squares for treatmentsMSE refers to mean sum of squares for errors

Endnotes

Tortora, ct.al., "Microbiology An Introduction". Six Edition. 2002. pp. 172-173.Mendenhall. mat., "Introduction to Probability and Statistics", Tenth Edition. pp.722-724

Chapter 4"GOLDEN KUHOL"

(Pomacea canaliculata)

AS SUBSTITUTE PROTEINSOURCE IN THE CULTURE OF

 Escherichia coil 

This chapter presents the presentation of analysis and interpretation of datathrough tabular and textual form.The analysis, presentation and interpretation of data were based on the number of colonies produced in each culture media. This variable would determine whether there isa significant difference between the effects of commercial nutrient Agar medium and"Golden Kuhol" medium as source of protein in bacterial growth

Effect of the Substrate Using "Golden kuhol" as Protein Source in Growing of Escherichia coli Bacteria as shown by the number of colony growth.Table INumber of Bacterial Colonies Crown in Dilution 104]TREATMENTNO.

showed the average number of colonies observed on the culture mediumafter 48 hours in dilution 10

-5

. The data revealed that treatment 1 (control) attained thehighest average number of colonies which is 333. Treatment 2 (kuhol media) attained 295average numbers of colonies next to the control treatment, which makes it an alternative protein source in culturing bacteria. The data implied that golden kuhol as protein source

hastens the growth of bacteria. Treatment 3 attained no growth of bacteria.

Significant Difference Between the Effect of Commercial Nutrient Agar and GoldenKuhol Medium as a Source of Protein in Bacterial Growth.

Statistical analysis using ANOVA revealed that the computed F-value of 4.4 isless than the tabular F-value of 7.56 at 11 level of significance, thus null hypothesis isaccepted. This further revealed that there is no significant difference between the effect&commercial protein agar and "golden kuhol" medium as a source of protein in bacterialgrowth

Chapter 5SUMMARY, CONCLUSIONS AND RECOMMENDATION

This chapter contains the summary, conclusions and recommendations which were formed from the data presented in the previous chapter.

Summary

This primarily aimed to utilize "golden kuhol" as an alternative protein source tocreate a non-synthetic medium for bacterial growth. The objectives of this study were:1.What is the effect of the substrate using "Golden Kuhol" as protein source ingrowing

 Eveherichia coli

 bacteria as shown by the number of colony growth?2. Is there any significant difference between the effects of commercial nutrientagar and "Golden Kuhol" medium as source of protein in bacterial growth?The null hypothesis stated that there is no significant difference between theeffects of commercial nutrient Agar and "Golden Kuhol" medium as source of protein in bacterial growth.This study used Randomized Complete Block Design (RCBD) with three (3)treatments replicated six (6) times The data were interpreted using Analysis of Variance(ANOVA) as appropriate tool.

Findings

Based from the gathered data, the following findings were revealed:1.The results showed that treatment I (control) attained the highest number of colonies which is 333 after 

L

IS hours of incubation Treatment 2 (kuhol agar) attained the

average number of 295 colonies, which is not far from the control treatment. Treatment 3(Agar-Agar) attained no growth at all.2. Statistical analysis using ANOVA revealed that the computed F-value of 4.4 isless than the tabular F

.

-value of 7.56 at I% level of significance

;

thus null hypothesis isaccepted. This further revealed that there is no significant difference between the effectsof commercial protein Agar and "Golden Kuhol" medium as source of protein in bacterial growth.

Conclusions

Based on the findings of this study, the researchers concluded the following:1. "Golden kuhol"

(Pomacea canalikuta)

is an effective protein source in growing

 Escherichia coli

as shown by the number of colony growth.2.The is no significant difference between the effects of commercial protein Agar and "Golden Kuhol" medium as source of protein in bacterial growth.RecommendationsIn view of the above findings and conclusions

:

the following recommendationswere formulated:1. It is recommended that further research be conducted for other possible protein sources for the protection of a bacterial culture medium.2.Other researches can be undertaken to find other ways of utilizing kuhol.3. It is also recommended that other researches he conducted for other substitutes to beef as a source of protein in culturing bacteria.

EndnotesBOOKS

Fernandez, W.L. et.al., "General Microbiology". 1995. 2

'

edition. Institute of Biological Science, UPLB.Quimio, A.J. and T.H. Quimio, 2001. “Introduction to Fungal Pathogens".University of the Philippines, Los Banos Laguna. College of Agriculture PublicationOffice.Tortora, et.al., "Microbiology An Introduction". Sixth Edition. 2002. pp. 172-173.Case Study of Golden Apple Snail, U.S. Department of State. 2003. pp.14-16.Rice Technology Bulletin. 2001. No.33Mendenhall, et.al., "Introduction to Probability and Statistics", Tenth Edition. pp.722-724.

UNPUBLISHED THESIS

Farolan, et.al., "Pulverized Golden Kuhol as Protein Source in CulturingBacteria", Unpublished Undergraduate Thesis, Ateneo de Naga. 2000.Hernandez, et.al., "Different Concentration of Ginger Extract In Eseherichia coli",Unpublished Undergraduate Thesis, CNSC, Daet, Camarines None.