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    Procedure Procedure Document Transcript

    • Sampling Procedure Since the research is all about the potential of Golden Apple Snail meat as a culture medium nutrient source, the researchers only chose this kind of snail as this is way easier to obtain and usually found in rice fields. Hence, the Golden Apple Snail served as the representative for the experiment. Research Procedure Before the experiment will be conducted, the researchers will secure the permission of their Program Dean to begin. They will send letters to the above-mentioned sources, asking for the mentioned microorganism. Guided and monitored by their thesis adviser, the researchers will then be conducting the experimentations. They will do trial-and-error experiments in preparing culture media from the snail meat to come up with the optimum (based on texture, appearances, and consistency) formulation or preparation to be used in the subsequent microbial growth testing. The overall and detailed procedures are presented below: 1. Overall methodology Before the researchers will come up to the final product, which is the Golden Apple Snail Culture Medium, they will take significant steps and procedures. First, they will gather the snail by obtaining them from a rice field. Then, they will prepare the snail meat as a nutrient source for culture medium. The researchers will extract the snail meat concentrate by separating the shell from the meat and will process it using a blender. The snail meat powder will be made by drying the snail meat in an oven for how many days and processed it under a grinder. After the extraction, the researchers will then prepare the culture medium using snail meat as a nutrient source. For the solid and semi-solid culture medium, they will add the snail meat powder of different formulation with the specific amount of agar powder, dissolved in distilled water and heated in a hot plate for a few minutes. The positive controls, which were the Nutrient agar and Sulfide Indole Motility medium, will be prepared but only with the addition of distilled water and undergone heating process. Then, the solution will be sterilized, poured in a plate respectively and allowed to solidify and cooled. The organisms will be allowed to grow by streaking and stabbing in the medium and later be observed for growth after a specified time of incubation.
    • Overall Methodology Figure 2. Flow Chart of Overall Methodology II. Preparation of the snail meat powder and concentrate When the Golden Apple Snail will be obtained, the researchers will process the meat for incorporation of the appropriate form of snail to the medium. They will make the concentration by separating the shell from the whole meat, sliced in small bits and blended in a blender. Then, they will extract the liquid by squeezing with a cloth and will be filtered in a flask. For the snail meat powder, the whole meat will be sliced thinly. Then, they will pile them in a tray and will be allowed to dry in an oven for almost a week. When the snail meat is dry, they will be grinded to produce snail meat powder. GATHERING OF GOLDEN APPLE SNAIL MAKING SNAIL MEAT NUTRIENT SOURCE FOR CULTURE MEDIUM PREPARATION OF CULTURE MEDIUM USING GOLDEN APPLE SNAIL MEAT AS NUTRIENT SOURCE PREPARATION OF CULTURE MEDIA USING NA/SIM/NB AS POSITIVE CONTROL SOLIDSOLID SEMI-SOLID SEMI-SOLID GROWTH OF MICROORGANISMS
    • III. Qualitative and quantitative tests for macro- and micronutrient content of the snail meat powder and concentrate prepared in part II above. 1. Molisch test is a general test for all carbohydrates and any compound containing a carbohydrate residue in the molecule. The starch solution would serve as a positive control. See figure below. Figure 5. Molisch Test The Benedict’s Test is a test for glucose present in the banana. See figure below. Figure 6. Benedict’s Test Place 1ml snail meat extract in a tube Add 1 drop of Molisch reagent. Mix. Incline the tube and aloow 1ml of conc. H2SO4 to flow at the side of the tube. Note thecolor produced at the juncture. 1ml Benedict’s solution + 2 drops snail meat extract. Boil for 2 minutes and allow to cool. Observe for the formation of the brick red precipitate which indicates presence of glucose.
    • The snail meat extract can also be tested for the presence of protein or amino acid. The test will be the Biuret test and the positive control will be 3-6% dilute ovalbumin solution. See figure below. Figure 7. Biuret test IV. Formulation of solid, semi-solid, and liquid snail meat culture media using the banana powder and concentrate prepared in part III above. The formulation was based on the composition of the commercial nutrient agar and sulfideindole motility. The following were the different formulations made for the experimentation: For solid medium: 8g snail meat powder + 15g agar powder in 1000 ml dis. water 12g snail meat powder + 15g agar powder in 1000 ml dis. water 16g snail meat powder + 15g agar powder in 1000 ml dis. water For semi-solid medium 8g snail meat powder + 7.5g agar powder in 1000 ml dis. water 12g snail meat powder + 7.5g agar powder in 1000 ml dis. water 16g snail meat powder + 7.5g agar powder in 1000 ml dis. water 1ml of snail meat extract and 1ml NaOH Add 0.5% of CuSO4 dropwise mixing thoiroughly after each addition. Observe Violet color = presence of long chain proteins Pink color = presence of short chain proteins
    • We made these formulations since they gave the most noticeable growth among all the formation tested. Figure 8. Formulation of solid and semi-solid snail meat culture media V. Preparation of the nutrient agar and Sulfideindole motility as positive controls for solid and semi- solid respectively. In preparing the nutrient agar solid media, first, wash two 1 L media bottles and black caps. Label one bottle. Wash one 1L beaker. Measure the dehydrated agar base required to make 1L of medium per the manufacturer’s printed instructions. Place the powder into the beaker. Very slowly, add 800 ml of distilled water to the beaker, running the liquid down the side of the vessel. Place the beaker on the hot plate stirrer. Turn on the stirrer on the hot plate (to lo) and allow the solution to mix. Turn on the hot plate, heating the solution at medium. Do NOT allow the solution to boil. After heating, using the hot hands protectors, remove the beaker from the hot plate and pour the solution into the labelled 1L media bottles. Fill each bottle to half of its capacity. Cap the bottles but ensure that the caps are loose but do not easily come off. Place the bottles into the autoclave and run them at 15-20 minutes. Cool the agar to 65 degrees Celsius. Make a formation using varying amount of snail meat and same amount of agar for solid (15g) and semi-solid (7.5) Mix the snail meat powder and agar in 1000ml distilled water Set the mixture into boiling with constant stirring until solutes are dissolved Sterilize, pour into plates (for solid) and tubes and allow to solidify.
    • Figure 9. Preparation of Nutrient Agar Solid Medium as a Positive Control For the semi-solid positive control medium, Scharlau Sulfide Indole Motility (SIM) medium was prepared using the following procedure: 1. Suspend 22g of the medium in one liter of purified water 2. Heat with frequent agitation and boil for one minute to completely dissolve the medium. 3. Autoclave at 121o C for 15 minutes Wash 1L beaker Measure agar based on manufacturer’s insert Place powder into the beaker Add distilled water Place the beaker on a hot plate. Stir Do not allow to boil Remove from the hot plate and pour into plates Suspend 22g of powder in 1L of distilled water waterwater Boil for 1 minute to dissolve the medium Autoclave at 121o C for 15 minutes and allow to cool
    • Figure 10: Preparation of SIM Medium as Semi-Solid Positive Control VI. Testing for growth of the selected microorganisms on the Snail meat media prepare in part IV above vs. nutrient agar and SIM as positive control. The following are the procedure on how to test for selected microorganism on the solid snail meat culture medium. Sterilize your work surface before beginning. 1. Obtain a petri plate of nutrient agar. 2. Flame- sterilze an inoculating loop, cool it on a spot of uncontaminated agar, and collect a colony of S. aureus and E. coli from stock broth culture. 3. Streak the bacterial colony onto a sterile agar Petri plate using the “triple Z “method. 4. Repeat for all the other plates, ensuring to sterilize the inoculating loop between plates. 5. Report the results based on the degree of growth (25%, 50%, 75%, 100%) Obtain a Petri plate of Nutrient agar Flame-sterile an inoculating loop, cool and collect a colony of organism from the broth culture. Streak the bacterial colony onto the agar plate. Repeat for all the other plates and report he result.
    • Figure 12. Testing for growth of selected microorganism is a Solid Snail meat Culture Medium For testing of selected organisms in semi-solid, the procedures were 1. Prepare a snail meat culture medium. 2. Flame-sterilize a wire needle and collect a colony of organism from the broth culture. 3. Inoculate the organism in the medium by stabbing and cover the tube with a sterilized cotton 4. Repeat the procedures 1 and 3 with other tube. 5. Incubate the tubes at 35-37o C according to the required time. 6. Report the result according to the degree of growth. Figure 13. Testing of Selected Organisms for growth in a Semi-Solid Medium Obtain a Semi-Solid medium Flame-sterilize an inoculating needle, cool and collect a colony of organism from the nutrient Stab the bacterial colony on the tube Repeat for all the other tube Incubate the tube at 35-37o C in a required time Report the result according to the degree of growth
    • These will be the procedures or testing the growth of selected organisms in solid snail meat culture medium: 1. Prepare a Snail Meat Culture medium. 2. Flame-steriize a wire loop and collect a colony from a pure culture of organism grown in nutrient agar. 3. Inoculate the colony by stirring the loop in the solid medium 4. Repeat procedures 1 to 3 for all the tubes. 5. Cover the tube and incubate at 35-37o C according to the required time. 6. Read and record the results according to the degree of growth. 7. The tube with the most noticeable growth was then tested further for confirmation by replanting it in a semi-solid medium. 8. Read and record the result of the semi-solid medium. Obtain the Snail meat culture medium Flame-sterilize an inoculating loop, cool and collect a colon in a nutrient agar Inoculate the bacterial colony other tube by stirring Repeat for all the other tubes Incubate the tubes at 35-37o C in a required time. rePort the result according to the degree of growth Test the tube with most prominent growth for confirmatory Read and record the results according to degree of growth