1. 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.

2. 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

3. Figure 3. Flow Chart of Making Snail Meat Concentrate
Figure 4. Flow chart of Making Snail Meat Powder
SEPARATE MEAT FROM SHELL
SLICE IN SMALL BITS
BLEND
EXTRACT THE LIQUID
STERILIZED BY BOILING
SEPARATE MEAT FROM SHELL
SLICE THINLY
DESSICATE OR DRY IN AN OVEN
GRIND

4. 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.

5. 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

6. 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.

7. 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

8. 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.

9. 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

10. 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