Ramon Magsaysay Technological University

San Marcelino Campus

San Marcelino, Zambales

NARRATIVE REPORT

in

MUSHROOM TISSUE CULTURE

Practicum in Agriculture

Presented to the Faculty of

College of Agriculture and Veterinary Medicine

In Partial Fulfillment of the Requirements

for the Degree in

Bachelor of Agricultural Technology

by

SHERILYN YAP CATE

DENNIS OLIVA GARCIA

DECEMBER 2018
 Ramon Magsaysay Technological University
San Marcelino Campus
San Marcelino, Zambales

APPROVAL SHEET

This narrative report entitled “Mushroom Tissue Culture” prepared and

submitted by Sherilyn Y. Cate and Dennis O. Garcia in partial fulfillment of the

requirement for the degree in Bachelor of Agricultural Technology had been reviewed and

recommended for acceptance and presentation.

PROF. MILA M. PATRIANA

Practicum Adviser

__________________________________________________________________

EVALUATION COMMITTEE

MS. NINA EZYLA PATRIANA PROF. DAVID P. PILIEN

Member Member

DR. MA. ESTER DLR. MARIÑAS

Chairman
 TABLE OF CONTENTS

Contents Page

APPROVAL SHEET i

LIST OF TABLES iii

LIST OF FIGURES iv

ACKNOWLEDGEMENT 1-2

BIOGRAPHICAL SKETCH 3

INTRODUCTION 4-5

Background Information 6
Objectives of the Project 7

PROGRAM OF WORK 7

SCOPE AND LIMITATION 9

Technical Performance 10

Production Process 10

Preparation of Culture Media 10

Potato Dextrose Agar Preparation 11
Tissue Culture Preparation 11

Sub culture Media Preparation 15

Sub culture Inoculation Preparation 15

Marketing Performance 20

Product Description 20
Pricing 20
Product Distribution 20

Financial Performance 21

Source of Fund 21
Total Project Cost 21
Cost and Return Analysis 22

PROBLEMS AND RECOMMENDATIONS 25

 LIST OF TABLES

Table Page

1 Work Plan and Timetable of Activities 9

2 Expenses of Pure Culture 22

3 Expenses of Sub-Culture 23

4 Gross Sale of Pure Culture and Sub-Culture 24

iii
 LIST OF FIGURES

Figure Page

1 Peeling and slicing the potatoes 12

2 Boiling of potatoes 12

3 Adding agar and sugar in decoction 13

4 Dispensing 50 ml in each flat bottle 13

5 Sterilization of medium for 2 hours 14

6 Inoculation 14

7 Washing the cracked corn in distilled water with Clorox solution 16

8 Boiling the cracked corn 16

9 Draining water from the cracked corn 17

10 Weighing the cracked corn 17

11 Sterilization of sub culture media 18

12 Inoculation process 18

13 Incubation process 19

iv
 ACKNOWLEDGEMENT

The student-trainees would like to express their sincere thanks and deepest gratitude

to all people who spent their time, provided support and guidance in any possible way to

make this narrative report completed and possible.

To the University President, Dr. Cornelio C. Garcia and Campus Director, Dr.

Nestor Z. Rondina of Ramon Magsaysay Technological University (RMTU) - San

Marcelino Campus for leading the university towards the center of excellence in

Agricultural Education in the province of Zambales.

To Prof. Mila M. Patriana for her support and assistance and time devoted for this

project and narrative report as their practicum adviser.

To Dr. Ma. Ester Mariñas, Ms.Niña Ezyla M. Patriana, and Prof.David P.

Pilien for their effort and guidance, for sharing their time, and for giving the student-

trainees useful, helpful, and sensible advices.

To their friends and classmates, for all the unforgettable memories and meaningful

ideas that they have shared together. The student-trainees are thankful to all of you.

To their beloved PARENTS and GUARDIANS, who were their inspirations for

being supportive in making this project successful and for the care, love, patience, and

understanding extended to them. The student-trainees are very thankful from the bottom of

their heart.
 Above all, to God the Father Almighty, for the countless blessings, knowledge,

strength, love, and guidance in preparing this project and writing the narrative report. The

student-trainees are humbly returning back all the glory in the name of Jesus Christ.

2
 BIOGRAPHICAL SKETCH

Dennis Oliva Garcia was born on December 2, 1998. He is the 7th child among the

8 children of Mr. Dominador Garcia and Mrs. Lolita O. Garcia. He grew up in Barangay

Dalanawan in San Marcelino, Zambales from where he finished his elementary education

at Dalanawan Elementary School in 2011. He continued his secondary education at San

Marcelino National High School in San Marcelino, Zambales.

At present, he is taking up Bachelor in Agricultural Technology (BAT) at the

Ramon Magsaysay Technological University-San Marcelino Campus. He is now residing

in Barangay San Rafael of the same town, San Marcelino in the province of Zambales.

BIOGRAPHICAL SKETCH

Sherilyn Yap Cate was born on August 20, 1998 in Caloocan City. She is the

daughter of Mr. Gilberto Cate, Jr. and Mrs. Sheryl Yap and also the second child among

the five siblings in the family. She grew up in Caloocan City and presently residing in

Barangay Patrocinio in San Narciso, Zambales. She finished her elementary education at

San Jose Patrocinio Elementary School in 201.1 She continued and finished her secondary

education in 2015 at San Antonio National High School (SANHS) in San Antonio,

Zambales.

She is now in her third-year level taking up Bachelor in Agricultural Technology

(BAT) at the Ramon Magsaysay Technological University (RMTU) - San Marcelino

Campus, San Marcelino, Zambales.

 INTRODUCTION

Importance of the Project

Mushrooms belong to Kingdom Fungi - class Basidiomycotan. These are basically

heterdraphicoraganisms mycelium of which thrives on waste materialcontaining cellulose

and pectic material.

The activities of mycelium during its course of life produce fruiting bodies which

are called mushrooms. They are rich in protein, carbohydrates, lipids, minerals, vitamins,

and contain certain biochemical and antibiotics.

Mushrooms are in demand because of their rich nutrition value and biochemical

value. These help to cure diseases like malnutrition, cardiac disorders, diabetes, cancers,

ulcers and other ailments. They are also used as health supplements because of high protein

content and high absorbability.

Mushroom culture is one of the simplest applications of biotechnology. Mushrooms

are fungal origin. They have universal focus and importance because of their nutrition

value and therapeutic value which can be used for remedy of various diseases and

disorders.

About 2500 varieties of mushrooms are available. Out of which varieties about 200

are edible and 12 varieties are commercially produced in India. Due to their commercial

viability and scientific importance, studying and learning mushroom culture has become

very important and interesting in India. In addition, this activity can generate self-

employment opportunities in urban and rural areas.

 The project served as a venue for DAT-BAT students to perform and master the

basic skills in mushroom tissue culture practices.

5
 BACKGROUND INFORMATION

The project Mushroom Tissue Culture dealt with the establishment of mushroom

tissue culture project within the College of Agriculture and Veterinary Medicine (CAVM)

at Ramon Magsaysay Technological University- San Marcelino Campus.

It was conceived primarily to support instruction and as production venture in

addition to the existing production projects of the university. As a support to instruction,

the project serves as a venue for DAT-BAT students to perform and master the basic skill

in mushroom tissue culture practices. As a production venture, it is considered as

entrepreneurial in nature and characteristics. And with this, the project aimed at producing

and selling pure culture and sub culture.

OBJECTIVES OF THE PROJECT

General Objective

This project was undertaken primarily to establish a narrative report on Mushroom

Tissue Culture to be presented to the faculty of College of Agriculture and Veterinary

Medicine (CAVM) at the Ramon Magsaysay Technological University which served as a

training ground for DAT-BAT students.

Specific Objectives

1. To serve as an income generating project of the student-trainees and of the school.

2. To supply the pure culture and sub culture needs of rural farmers.

PROGRAM OF WORK

This project started its implementation on November 16, 2016.

Table 1 shows the work plan and timetable of activities of mushroom tissue culture

at Ramon Magsaysay Technological University- San Marcelino Campus. These included

the purchase of procurement of supplies and materials.

Table 1. Mushroom Tissue Culture Work Plan and Timetable

 DATE ACTIVITY RESOURCE
REQUIREMENTS
Nov.16-18, 2016 Procurement of supplies, See attachment
equipment and materials.
Nov. 21, 2016 Preparation of the laboratory Labor
room.
Casserole, measuring cup,
Nov. 23-24, 2016 weighing scale, stove, flat
Preparation of potato dextrose bottles, aluminum foil, cotton,
agar (F0) rubber band, gulaman bars,
sugar, potato, cheese cloth,
and pressure cooker
PDA slants, wire needle,
alcohol lamp, rubbing alcohol,
Nov. 25-29 Isolation and incubation of denatured alcohol, match,
pure culture (F0) inoculating chamber, and pure
culture of Pleurotusostreatus
Casserole, weighing scale,
cracked corn, bleach, round
Dec. 5, 2016 Preparation of sub culture (F1) bottles/polypropylene plastic
bag, alumina foil, stove,
cotton, and rubber band
Prepared media, inoculating
Inoculation and incubation of rod, alcohol lamp, rubbing
Dec.06-09, 2016 sub culture (F1) alcohol, denatured alcohol,
match, inoculating chamber,
and pure culture media
Casserole, weighing scale,
bleach, cracked corn,
Dec. 13, 2016 F2 Preparation polypropylene plastic bag,
aluminum foil, cotton, rubber
band, stove, and F1 culture
stock.
Prepared media, inoculating
rod, alcohol lamp, rubbing
Dec. 14-17, 2016 Inoculation and incubation of alcohol, denatured alcohol,
sub culture (F2) match, inoculating chamber,
and sub culture media (F1)
Dec .20, 2016 Preparation of sub culture (F2) See attachment

Dec .21-24, 2016 Inoculation and incubation of See attachment

sub culture (F2)

SCOPE AND LIMITATION

 The project Mushroom Tissue Culture was initiated with the following

limitations:

a. The mushroom tissue culture was limited to 5 kilograms of cracked cornor 30

polypropylene bags (4 x 9 inches) weighing 200 grams.

b. The time frame of its initial implementation was November 16, 2016 to April of

2017. The activities within the time frame included the setting up of the equipment.

c. DAT-BAT students were utilized in the conduct of the project as their practicum

in Agriculture.

TECHNICAL PERFORMANCE
Production Processes

A small laboratory was provided and utilized as the laboratory room where cultures

of Pleurotus ostreatus were maintained. Thirty polypropylene bags and fourteen flat

bottles of Pleurotus ostreatus were produced as initial production.

The pure culture and subculture were sold to farmers. From the 30 polypropylene

bags of subculture produced, 9 bags were contaminated and from the 14 flat bottles of pure

culture produced, 3 were contaminated.

Preparation of Culture Media

The materials in preparation of culture media are gulaman bar, sugar, potato,

measuring cup, blade/scalpel, cotton, aluminum foil/paper, rubber band, beaker, weighing

scale, cheesecloth, and distilled water.

10

Potato Dextrose Agar Preparation

 Preparing the potato dextrose agar followed different steps. First, the student-

trainees washed, peeled, and diced the potatoes. (Figure 1). Potatoes weighing 800 grams

were placed in a casserole. (Figure 2). Second, the student-trainees strained the broth

through the cheese cloth. They restored the volume of decoction to 1L and put back into

the casserole. Third, they added the white gulaman bar and poured the white refined sugar.

They heated and stirred it until the gulaman bar get dissolved. (Figure 3). Then, they

dispensed 50Ml in each flat bottle and plugged the mouth of the bottle with cotton to avoid

contamination. (Figure 4). They sterilized the medium in a casserole for two (2) hours.

(Figure 5). After sterilization, they lay the bottles flat on the table until the agar congealed.

Then they let it cooled and solidified. (Figure 6).

Tissue Culture Preparation

Rubbing alcohol with 70% alcohol content was prepared. This served as a

disinfectant.

First, cut the mushroom into thin pieces and dipped the tissue in the container where

the disinfectant was placed. Then dipped the tissues with disinfectant together with a

distilled water. The tissue was picked one at a time with the use of sterilized inoculated

needle and planted them on the flat bottle (Figure 7). Set aside for incubation.
Figure 1. Peeling and Slicing of Potatoes

Figure 2. Boiling of Potatoes

12
Figure 3. Adding the Agar and Sugar in Decoction

Figure
4.

Dispensing 50ml in Flat Bottles

13
 Figure 5. Sterilization

Figure 6. Inoculation Process

14

Sub Culture Media Preparation

 The materials used in the preparation of sub culture media were cracked corn,

water, Clorox solution, polypropylene plastic bag/round bottles, aluminum foil/paper,

cotton, weighing scale, PVC pipe, and 70% rubbing alcohol.

First, cracked corn was washed in distilled water with Clorox solution to eliminate

the presence of contaminants. Then washed it again in distilled water without Clorox

solution. (Figure 7). After washing the cracked corn, they boiled the cracked corn in a

casserole until the first boil or the cracked corn can be pinched. The student-trainees cooled

down and drained it. (Figure8-9). Cracked corn was packed using polypropylene plastic

bags (4 x 9) weighing 200 grams (Figure10). Prepared media was sterilized for about 2

Hours (Figure11). Inoculation process. (Figure 12). Incubation process. (Figure 13).

Sub Culture Inoculation Preparation

The materials needed including the room and the inoculating chamber in sub culture

inoculation preparation were disinfected using water with Clorox solution and alcohol.

This way would eliminate contaminants. All the materials needed in the inoculation were

placed inside the inoculating chamber. A solution that served as disinfectant was prepared.

Then 1 x 1 cm of pure culture was lifted and transferred into sterilized prepared media.

After transferring the pure culture, enclosed it again to avoid the entrance of

microorganisms.
Figure 7. Washing the cracked corn in distilled water with Clorox solution

Figure
8.
Boiling
the
cracked
corn

16
Figure 9. Draining water from the cracked corn

Figure 10. Weighing the cracked corn

17
Figure 11. Sterilization of Sub Culture Media

Figure 12. Inoculation Process

18
Figure 13. Incubation Process

19
 MARKETING PERFORMANCE

Product Description

The project had two (2) products called pure culture and sub culture.

The pure culture was produced in potato dextrose agar measuring 50 ml. It was

inoculated with tissue of fresh oyster mushroom, plugged with cotton and covered with

aluminum foil/paper.

The sub culture was produced in cracked corn. The weight of sub culture was 200

grams in every round bottle or polypropylene plastic bag and it was inoculated with pure

culture.

Pricing
 The pure culture was sold at ₱300.00 per bottle while the sub culture was sold at

₱100.00 per 200 grams per bottle or polypropylene bag (4 x 9).

Product Distribution

Pure culture and Sub culture were sold to the local community. Most of the

consumers were students and farmers.

FINANCIAL PERFORMANCE

Source of Fund

The materials and supplies were provided by the Department of Agriculture and

support from student-trainees’ parents, guardians, and friends who conducted the

mushroom tissue culture.

A total of ₱1,987.79 for pure culture and ₱2,030.16 for sub culture have been

incurred.

Total Project Cost

The total project cost incurred in establishing this mushroom tissue culture

project was ₱4,017.95.

 21

Cost and Return Analysis

Table 2. The table shows all the expenses for Pure Culture

A. Laboratory Supply

Pure Culture

PARTICULAR UNIT QUANTITY UNIT COST TOTAL

COST
Gulaman bar Bar 8 ₱ 12.50 ₱100.00
Potato Gram 800 0.1 ₱ 80.00
Sugar Gram 80 0.048 ₱ 3.84
Distilled water Liter 4 ₱ 22.00 ₱ 88.00
Cotton Gram 100 0.28 ₱ 28.00
Rubber band Piece 14 0.16 ₱ 2.24
Mushroom Gram 125 0.20 ₱ 25.00

SUB TOTAL ₱327.08

B. Labor

PARTICULAR UNIT QUANTITY UNIT COST TOTAL

COST
Preparation Man, per day 2 ₱ 200.00 ₱ 400.00
Sterilization Man, per day 2 ₱ 200.00 ₱ 400.00
Inoculation Man, per day 2 ₱ 200.00 ₱ 400.00
 SUB TOTAL ₱ 1,200.00

C. Transportation ₱ 280.00
D. Miscellaneous ₱180.71
TOTAL: ₱ 1,987.79

22

Table 3. The table shows all the expenses for Sub Culture

A. Laboratory Supply

Sub Culture

PARTICULAR UNIT QUANTITY UNIT COST TOTAL

COST
Cracked corn Grams 5000 0.026 ₱130.00
Clorox Solution Ml 250 0.048 ₱ 12.00
Cotton Grams 200 0.28 ₱ 56.00
Rubber Band Piece 60 0.16 ₱ 9.60
Foil Roll 2 ₱ 25.00 ₱ 50.00
Polypropylene Piece 30 0.70 ₱ 21.00
Plastic Bag
Denatured Ml 350 0.0714 ₱ 25.00
Alcohol
70%Rubbing Ml 500 0.124 ₱ 62.00
Alcohol
SUB TOTAL: ₱365.60

B. Labor

LABOR UNIT QUANTITY UNIT COST TOTAL COST

Preparation Man, per day 2 ₱ P200 ₱400.00

 Sterilization Man, per day 2 ₱ P200 ₱400.00
Inoculation Man, per day 2 ₱ P200 ₱400.00
SUB TOTAL ₱1,200.00

C. Transportation ₱280.00
D. Miscellaneous ₱184.56
TOTAL: ₱2,030.16

23

GROSS SALE

Table 4. The table shows the Gross Sales of Pure Culture and Sub Culture

PURE CULTURE

MONTH UNIT QUANTITY UNIT COST TOTAL COST

December Bottle 11 ₱300.00 ₱3,300.00

SUB TOTAL ₱3,300.00

SUB CULTURE

MONTH UNIT QUANTITY UNIT COST TOTAL

COST
December Polypropylene 15 ₱100.00 ₱1500.00
Plastic Bag
January Bottle 6 ₱100.00 ₱600.00

SUB TOTAL ₱2,100.00

 TOTAL ₱5,400.00

GROSS INCOME: ₱ 5,400.00

TOTAL EXPENSES: ₱ 4,017.95

NET INCOME: ₱ 1,382.05

RETURN OF INVESTMENT: 34.40%

24

PROBLEMS AND RECOMMENDATION

Table 5. The table shows the problems encountered and recommendation.

PROBLEMS CAUSES SOLUTION

Media fails to solidify. Insufficient quantity of agar Thoroughly mix media
or distribution of thereof. before pouring.
Contamination occurs in High contaminant spore Clean, paint laboratory
petri dishes after pouring load in laboratory.
the media but before
inoculation. Improper media preparation Allow pressure cooker to
technique. cool in sterile setting before
opening.

No growth from spores or Wrong type of media. See media preparation.

tissue transferred.
Scalpel or loop too hot. Cool tool before contacting
spores or tissue
Contamination occurs Inoculum contaminated. Take a tissue culture from a
around point of transfer fresher specimen.
onto agar media. Inoculation tools not sterile. Inoculation tools, soak in
alcohol, flame, sterilize
before using.
25