INTRODUCTION

A. Background of the Study

Even with the advancement of technology we have today which

improves the agriculture sector of the Philippines, there are still factors that

challenge its manufacture. The inevitable conversion of agricultural land to

industrial land greatly affects the production of crops which leads to shortage

of domestic supply. According to Laranio and Oliveira (2013), the solutions to

these problems were already developed, reinforcing the importance of

sustainable intensification of plant production.

The biofertilization of crops with plant-growth-promoting organisms is

currently considered as a healthy alternative to chemical fertilizer (Fraile et.al,

2012). And Rhizobia is an effective bacteria to plant growth which can be

found in the root nodules of makahiya plants. Rhizobia are bacterial

symbionts of legumes that fix atmospheric nitrogen in a process known as

biological nitrogen fixation (BNF). These are soil bacteria that are able to

provide macronutrient to plants (Alexandre et.al, 2013).

Since tomatoes are one of the economically very important vegetables

whose raw fruits are consumed but are also susceptible to plant diseases, it

will be used as an experimental unit to test the effectiveness of Rhizobial

inoculant the plant growth of non-leguminous plants. This study to utilize the

Rhizobia from makahiya plant nodules to be used as a microbacterial

inoculant to tomato plants.

 d. 10-7

e. 10-8
B. Statement of the Problem
f. 10-9

The main objective of the study is to determine the specific species of

Rhizobia bacteria present in the makahiya plant nodules. Specifically, it aims

to answer the following questions:

1. Which of the following dilutions has the most bacterial

isolates?

a. 10-4

b. 10-5

c. 10-6

2. Which among the species of Rhizobium displays similar

characteristics as the rhizobia bacteria present in makahiya

root nodules?

a. Rhizobium japonicum

b. Rhizobium leguminusarum

c. Rhizobium trifolii

C. Significance of the Study

This research study gives use for the makahiya plant nodules. Since

the number of this species is enormous, utilizing it as a microbial inoculant will

greatly help in aiding in the agricultural sector specially in enhancing the

quality and production of crops. Rhizobia is known to be nitrogen-fixing which

can be found in plant nodules. Knowing that a root nodule has a number of

bacteria species due to microbial diversity, the correct characterization and

identification of Rhizobia is necessary to ensure the effectiveness of makahiya

as inoculant to yields. Moreover, the process of characterization and culturing

will lead to its application on several crops wherein it will serve as a good bio

fertilizer for crop production. Hence, aiding and helpful to the agricultural

sector.

D. Scope and Limitations

This study focuses on the morphological characterization of rhizobia

bacteria present in the makahiya plant root nodules. The researchers did not

include the biochemical characteristics of the bacteria because of limited

resources to do such tests. The study is limited on the experimentation and

tests which was performed in Regional Science High School IX.

 METHODOLOGY

A. Research Locale

The study was conducted at Regional Science High School IX,

Malasiga, San Roque, Zamboanga City.

B. Research Design

The researchers will utilize the use of Randomized Block Design as the

research design of the study. There will be six set-ups with two trials each.

Six set-ups with a dilution ranging from 10-4 to 10-9.

C. General Procedure

Collection of Plant Nodules

Mimosa pudica (makahiya) plants were gathered at Malasiga, San

RoqueZamboanga City. The makahiya roots were carefully uprooted, and the

root systems were washed under running water to remove the adhesive soil

particles. The color of the nodules varies from brown to pink depending on the

state of pigment present in them. For the experiment, healthy, unbroken pink

nodules were selected.

Surface Sterilization of Root Nodules

After washing and rinsing the root nodules for three times, the nodules

were then immersed in 70% ethyl alcohol for a minute for it to be sterilized

and no other microorganism or foreign material will be present.

Sterilization of Materials

The sterilization of materials was done using an autoclave and heating

the materials above the boiling point. The autoclave was set to 15 psi. This

was to ensure that the materials were sterile and free from microorganisms or

bacteria before and after the experiment procedure was done.

Serial Dilution Technique

After sterilizing the root nodules, they were placed in a petri dish with

1ml of sterile distilled water and were macerated with a scalpel. Once all the

root nodules were opened and crushed, tenfold serial dilution of nodular

extract was prepared by taking 1g of nodular extract into 10 ml of sterile

distilled water and mixed well to get the nodular extract suspension. 1ml of

nodular extract suspension was diluted with 9 ml of sterile distilled water

making the dilution to 10-1. Another 1 ml from the 10 -1 dilution and then

distributed to the second test tube making it 10 -2 dilution and so on. The

dilution was made up to 10-9.

Streaking

A wire loop was passed through a flame of an alcohol lamp to sterilize

it. The wire loop was dipped into the diluted solution. On the other hand, the

petri dish with the culture media was heated around and just above the flame;

the petri plate was slightly open so that the wire loop that was dipped in the

solution could streak on the culture media. It was streaked in an s-manner,

not touching the any of the petri dish.

Incubating

After successfully streaking with each dilution solution, the petri plated

was sealed with masking tape to secure that no further contamination may

happen. Each petri plate was labeled and placed upside down inside the

incubator. The samples were incubated for three days.

Viewing of Isolates under the Microscope (Characterization)

After incubating, the isolates present were gathered, and viewed under

the microscope. Gram staining was the technique used. The researchers

used a blue food color, in replacement for methylene blue for the first step of

staining. Later on, iodide solution was added, and a combination of 75% ethyl

alcohol (70% solution) and 25% acetone for the decolorization agent.

D. Disposal Treatment

The study utilized the nodules of the makahiya plant which can be

found in its roots. The other parts of the plant were thrown into a compost pit,

which soon may be used as fertilizer. Disinfectants like the alcohol and zonrox

were reuse for cleaning the laboratory to be used.

 RESULTS

After incubation of three (3) days, the following were the results

observed after the experiment.

Table 1. Tallied Number of Isolates Identified in Each Sample

Samples
Sample 1 (Dilution 10-4)
Trial 1 No. of Trial 2 No. of
Isolates Isolates
Five Five
(5) 2 3 (5)
2 3 1
4 4

1 5

Sample 2 (Dilution 10-5)

Trial 1 No. of Trial 2 No. of
Isolates Isolates
Six (6) Seven
(7)
6 5 1
2
1 1 3 7
4 2
4
3 6
3 5
 Sample 3 (Dilution 10-6)
Trial 1 No. of Trial 2 No. of
Isolates Isolates
Nine Nine
(9) (9)
4 9
3 5 5
4
8 9
2 7 3 6
8
6 2
1 1 7

Sample 4 (Dilution 10-7)

Trial 1 No. of Trial 2 No. of
Isolates Isolates
Ten Eleven
(10) 3 (11)
4 5 10 11
4 10
3 5
6 9
16
2
2 7 7
1 8 9 8
1
 Sample 5 (Dilution 10-8)
Trial 1 No. of Trial 2 No. of
Isolates Isolates
Eleven Eleven
(11) 11 (11)
4 5 10
11
4 10 9
3 5 3
2 8
2
9 1
7
6
6 8
1
7
1

Sample 6 (Dilution 10-9)

Trial 1 No. of Trial 2 No. of
Isolates Isolates
Thirtee Fifteen
n (13) (15)
1 11
2 10 9
1
3 12
8
9
8 4 13 14 7 2
5
8 3
10 7 6 15 4
11 6 5

Table 1 shows the data on the number of isolates found on each

sample. In Sample 1 containing the 10 -4 dilution, it is seen that it has the least

number of isolates which is only five, both in Trial 1 and 2. It is also observed

that the bacterial colonies growing in Sample 1 is larger compared to the other

samples. On the other hand, in Sample 6 containing the 10 -9 dilution, there are

13 up to 15 isolates identified in Trial 1 and 2 respectively. The sample had

fewer bacterial colony identified. In the data above, it can be inferred that the

lower the dilution, the more isolates are visible.

A. Rhizobial Isolates Viewed Under the Microscope

The sample was viewed under an eyepiece of 15x magnification, and

under different objectives (LPO and HPO) shown in the figures below.

Figure 1. Under LPO (12x magnification)

The half left of the image illustrates

these large cells filled up by the bacteria. Note

that the nuclei are also enlarged and even the

nucleoli (enveloped by a halo) are prominent.

On the half right of the image, vacuolated

plants cells were present. This image was

Figure 2. Under HPO (40x magnification)
obtained by having a magnification of 180x.

paint

Figure 2. The details of the specimen are very much clearer and

distinct, showing the details of the Rhizobium bacteria. This image was

obtained by having a magnification of 480x.

Table 2. Morphological Characterization of the Isolates

 Rhizobium sp.
CHARACTERISTICS Rhizobium Rhizobium Rhizobium Sample
japonicum leguminusarum trifolii X
1. Colony Shape + + + +
(Circular) (Circular) (Circular) (Circular)
2. Color ̶ + ̶ +
(Whitish (White) (Creamy (White)
pink) white)
3. Elevation ̶ + ̶ +
(Pulvinate) (Convex) (Flat) (Convex)
4. Surface + + ̶ +
Margin
(Entire) (Entire) (Ungulate) (Entire)
5. Opacity + + + +
(Opaque) (Opaque) (Opaque) (Opaque)
6. Motility + + + +
(Motile) (Motile) (Motile) (Motile)
7. Bacterium ̶ + ̶ +
Shape
(Rod (Circular) (Rod (Circular)
shaped) shaped)
8. Oxygen + + + +
Demand
(Aerobic) (Aerobic) (Aerobic) (Aerobic)
9. Gram’s + + + +
nature
(Gram- ve) (Gram- ve) (Gram- ve) (Gram-
ve)

Table 2 is a chart used which shows the morphological characterization

of the bacteria identified under the microscope. The data involves different

physical and cultural characteristics of a bacteria that served as a guide for

the sample characterization. In this case, three different types of rhizobia were

included to serve as a comparison to Sample X (rhizobium bacteria viewed

under the microscope). These three rhizobia species are Rhizobium

japonicum, Rhizobium leguminusarum, and Rhizobium trifolii. Based on the

observations, the characteristics of Rhizobium leguminusarum are all present

in Sample X.
DISCUSSION
 In the process known as Biological Nitrogen Fixation (BNF), Rhizobia

are bacterial symbionts of legumes that fix atmospheric nitrogen. The bacteria

in the soil are able to provide macronutrient to plants (Alexandre, 2013). The

proper isolation and characterization of Rhizobia species from Mimosa pudica

(makahiya) plant nodules has the potential of utilizing it as a microbial

inoculant. Based on the results of the study, as the dilution concentration

decreases, there is a greater number of isolates. The concentration of

bacteria was reduced in each dilution by a specific amount, hence, through

this method the pure culture of the Rhizobia may be obtained (Hartstock,

2016). Consequently, samples with lesser bacterial colony and more isolates

were the product of having greater level dilution. As the bacterial colonies

were successfully separated, it would be a lot easier to have pure cultures of

bacteria present in makahiya plant root nodules. In addition, samples with

greater dilution have lesser occurrence of contact resulting to have a lesser

chance of the samples being contaminated by foreign microorganisms.

In the process of viewing the bacteria under the microscope, Gram

staining was used. Gram staining is a common technique used to differentiate

two large groups of bacteria based on their different cell wall constituents. The

Gram stain procedure distinguishes between Gram positive and Gram

negative groups by coloring these cells red or violet. It involves three

processes: staining with a water-soluble dye called crystal violet,

decolorization, and counterstaining (Bruckner, 2016). Gram staining was also

used to be able to have a clearer and detailed vision of the bacteria’s

characteristics to be able to proceed in its characterization. In this case, the

researchers used a blue food color, in replacement for methylene blue for the
first step of staining. Later on, iodide solution was added, and a combination

of 75% ethyl alcohol (70% solution) and 25% acetone for the decolorization

agent. Gram staining was successful, having a clear and detailed image of the

Rhizobia bacteria. It was observed that there were a lot of bacteria cells

present which have distinct features and characteristics. This only shows that

the process of isolation and staining were successful. Furthermore, the gram

staining resulted to the Rhizobia bacteria belonging to a gram-negative group.

As part of the research objective, the researchers proceeded with the

characterization of the viewed bacteria. Morphological Characterization of the

bacteria was done. It is describing the cultural characteristics of the bacterial

colony and its cells, and later on be compared to the characteristics of

Rhizobia species which are already studied, like the Rhizobium japonicum,

Rhizobium leguminusarum, and Rhizobium trifolii Moreover, the bacteria

observed under the miscroscope (Sample X) has all the characteristics which

are also present in the Rhizobium leguminusarum. It only implies that the

rhizobia bacteria present in the root nodules of makahiya plant is identified as

Rhizobium leguminusarum.

CONCLUSION
 Based from the findings of the experiment, it can be concluded that

serial dilution technique can decrease the population of bacterial colony

producing numerous number of isolates. The dilution 10 -9 had the most

number of isolates present and visible. The higher the level of dilution of the

sample is, the lesser the population of bacterial colony, thus, making it easier

to have pure cultures of bacteria present in makahiya plant root nodules. In

addition, samples with greater dilution means that there is less occurrence of

contact resulting to have a lesser chance of the samples being contaminated

by foreign microorganisms. On the other hand, the rhizobium bacteria isolated

had characteristics of circular bacterial colony shape, white in color, convex

elevation, has an entire surface margin, opaque, motile and is also circular

when it comes to bacterial shape, it is also aerobic and proved to be gram

negative. These characteristics are identified to be present in Rhizobium

leguminusarum. Therefore, it is concluded that the Rhizobia bacteria present

in makahiya plant nodules is specifically identified and confirmed as

Rhizobium leguminusarum. These properties suggest that rhizobium isolated

in this study could find potential application for development of the sustainable

agriculture as to be a good candidate of biofertilizer which help in soil

fertilization without applying chemical fertilizers.

 RECOMMENDATIONS

Based from the results and analyses of the study, the researchers

made the following recommendations:

1. The researchers recommend utilizing the isolation of Rhizobium

leguminosarum from the Mimosa pudica (makahiya) plant nodules

as inoculants and to be applied in various crops such as rice,

wheat, cassava, etc. for its application to further study its effect on

the enhancement of the crops.

2. The researchers recommend using nodules of different leguminous

plants other than those from makahiya plants and subject them to

isolation of the Rhizobia bacteria.

3. It is suggested that the future researchers improve their process in

the isolation and utilize the most appropriate materials during the

experimentation.

4. The researchers recommend further research on the accuracy of

the isolation and the characterization of bacteria for a better

classification.

5. The researchers suggest to have a biochemical characterization of

Rhizobia bacteria present in the makahiya root nodules.

 ACKNOWLEDGMENT
The researchers wish to express their deepest gratitude and warmest

appreciation to the following people, who, in any way have contributed and

inspired the researchers to the overall success of the undertaking:

 Our beloved parents who have always been very understanding

and supporting us both financially and emotionally;

 Dr. Jocelyn Pedroso who continuously shared us her knowledge

and helped us in our entire Research Journey;

 The panel of judges who spent their time correcting the research

paper;

 Our understanding friends who have been extending their efforts to

help;

 To the school’s principal, faculty and staff for their support and

consideration;

 And above all, to the Almighty God, who never stops guiding us and

for the continued protection.

 APPENDICES

APPENDIX A: BUDGET AND TIME TABLE

BUDGET

Materials Cost (PHP)

Mimosa pudica (makahiya) nodules 0.00

Agar (20 grams) 35.00

Potatoes (500 grams) 50.00

Sucrose/Sugar (10 grams) 17.00

Distilled Water (6 Liters) 53.50

Graduated Cylinder 0.00

Test Tube 0.00

Surgical Gloves (2 pairs) 40.00

Wire Loops 18.00

Denatured Alcohol 30.00

Disinfectant - Ethyl Alcohol (150ml) 25.00

Disinfectant – Zonrox (100ml) 7.75

Cotton 63.50

Masking Tape 17.00

Cheesecloth (1/4 m) 11.00

Disposable Mask 0.00

 367.75
Subtotal

Others Cost (PHP)

Transportation Fee 700.00

Computer and Personal Cost

0.00

(Internet)
700.00
Subtotal

Report Reproduction Cost (PHP)

Computer time and Personal Cost

0.00
(Word Processing)

Communication 0.00

0.00
TOTAL

APPENDIX B: SCHEDULE OF ACTIVITIES

Activities Time Frame (in days)

A. Planning Stage

Choosing of the Study 5

Gather Information 10

Subtotal 15
 B. Implementation Stage

Combining Data 40

Encoding 5

Subtotal 45

C. Experimentation Data

Gathering of Materials 4

Experimentation 6

Observation 3

Subtotal 13

D. Analysis Stage

Analyze Data 7

Analyze the Research Report 4

Subtotal 11

E. Reporting Stage

Present findings at conference 1 day

TOTAL 84

APPENDIX C: DOCUMENTATIONS
Preparation of Potato-Agar
Dextrose
 The sterilization of materials
was done using an autoclave and
heating the materials above the
boiling point. The autoclave was set
to 15psi. This is to ensure that the
materials were sterile and free from
microorganisms or bacteria before
and after the experiment procedure
was done.

300 grams of potatoes was

cleaned and was chopped into little
cubes.

One (1) liter of distilled

water was poured together with
the potato cubes in a casserole
and was left to boil for 30
minutes.

The potato broth was

then filtered using the
cheesecloth and was set aside.
 A half kilo of agar was
washed and was chopped into
smaller pieces.

The chopped agar was

boiled until it was dissolved. The
dissolved agar was mixed with the
potato broth.

20g of sugar was added to

the potato-agar mixture and was
left to dissolve.

Surface Sterilization of
Root Nodules
 After washing and rinsing the
root nodule for three times, the
nodules were then immersed in 70%
ethyl alcohol for a minute so that it
would be sterilized, and no other
microorganism or foreign material
would be present.

After immersing the nodules

in an ethyl alcohol, the nodules
were then placed in another petri
dish added with 1mL distilled water
and was macerated.

Serial dilution technique

was done.
 Streaking

Streaking was done on the

media to make the bacteria culture
of decreasing dilution. It was
streaked in an s-manner, not
touching the any of the petri dish.

After successfully streaking

with each dilution solution, the petri
plated were sealed with masking tape
to secure that no further
contamination may happen. Each
petri plate was labelled.

Incubating for three days.

Viewing of isolates under the

microscope.
Viewing of Rhizobia isolates under
the microscope.
 REFERENCE LIST

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Rhizobium leguminosarum bv. viceae Strains Isolated from Different

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https://www.researchgate.net/publication/271196438_Diversity_of_Rhi

zobium_leguminosarum_bv_viceae_Strains_Isolated_from_Different_S

chemes_in_Shendi_Area?

fbclid=IwAR09E43g68J8Box4tGPZGJ1jEB4e9Qy61wn5IUCrS8fkDkqVi

YovMypS5Xk

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0474-8?fbclid=IwAR0_-

jmbr05HT90A1cO63VcZyHC5UiaQLZi6iX_Ew2dnp6bv_d6haJHmM7M

Alexandre G., Ramirez M., & Santos D.C. (2011). Research Gate . Retrieved

from Morphological, cultural and biochemical characteristics of

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https://www.researchgate.net/publication/304999423_Morphological_c

ultural_and_biochemical_characteristics_of_Rhizobium_japonicum_sy

n_and_Bradyrhizobium_japonicum_of_soybean?

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y2gjOrAi0MhIWmB662X70OhuE8gEKEVg_U
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Resources : http://serr.carleton

.edu/microbelife/research_methods/microspy/gramstain.html

Research Paper

Enriquez, J., Tulete , A., & Salih, I. (2017). DETERMINATION OF OPTIMUM

CONCENTRATION FOR ISOLATION OF Rhizobium sp. FROM

Mimosa pudica (makahiya) PLANT NODULES.

Journal

Enriquez, J., Tulete , A., & Salih, I. (2017). Research logbook.

DETERMINATION OF OPTIMUM CONCENTRATION FOR

ISOLATION OF Rhizobium sp. FROM Mimosa pudica (makahiya)

PLANT NODULES.