li report craun

INDUSTRIAL TRAINING REPORT

FOR

PRACTICAL SESSION

IN

CRAUN RESEARCH SDN. BHD.

Hassriana Fazilla binti Sapri

A 107251

Plant Biotechnology

School of Bioscience and Biotechnology

Universiti Kebangsaan Malaysia (UKM)

Hamizah binti Mokhtar

14094

Resource Biotechnology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak (UNIMAS)

CONTENTS PAGE

CHAPTER 1: CRAUN RESEARCH SDN. BHD.

1. Introduction 3

2. Mission 4

3. Vision 4

4. R & D Thrust Areas 4

5. Division

(a) Upstream Technology Division 5

(b) Downstream Technology Division 6

(c) R & D Commercialization Division 6

(d) Corporate Services Division 7

CHAPTER 2: INDUSTRIAL TRAINING PROJECT

1. Introduction 8

2. Materials and Methods 9

3. Schedule

4. Result

5. Discussions and Conclusion

6. References

CHAPTER 3: ADDITIONAL WORK

1. Literature Review

Cost Reduction via Alternative Gelling Agent in Plant

Tissue Culture

2. Medium Preparation Room Work

3. Others

CHAPTER 4: WORKLOAD FOR STUDENT WORK

APPENDIX

2

CHAPTER 1: CRAUN RESEARCH SDN. BHD.

INTRODUCTION

The Crop Research and Application Unit (CRAUN), of Land Custody and

Development Authority (PELITA) was established on 22nd July 1993 by the Sarawak

State Government. Upon the approval of the Sarawak State Government on April 1st ,

1997, CRAUN was corporatised and known as CRAUN Research Sdn. Bhd.

CRAUN began its operations at the Food Technology Research Laboratories of

the Malaysian Agriculture Research and Development Institute (MARDI), Sarawak

Branch on 15th February 1994. On 1st July 1994, the Farm Management and By-Product

Utilisation Section of CRAUN began its operation at the 1st and 2nd floor, Lot 500, Block

68, Mukah New Township, Mukah and on 17th September 1994 Sungai Talau Research

Station of the Agriculture Department Sarawak, was taken over by CRAUN. CRAUN’s

headquarters at Lot 3147, Block 14, Jalan Sultan Tengah, Kuching occupying

approximately seven acres, commenced construction on 17th November 1994 and began

its operations on 11th September 1995.

CRAUN Research Sdn. Bhd., is a Research and Development company,

specifically set up for the development of the Sago Crop and the Sago Starch Industry of

Malaysia and at the same time undertakes studies for the development of other crops that

have potential for commercial exploitation and application. CRAUN sets its vision to

achieve worldwide recognition as the authority on sago crop (Metroxylan sagu) and other

underexploited tropical crops of potential economic importance. The functions of

CRAUN have been arbitrarily divided into three main research divisions and a Corporate

Service Division.

3

MISSION

To generate and promote new improved technologies that increase the efficiency,

productivity and competitiveness of sago and sago-starch based industries in order to

assist the state to realize the transformation and modernization of the sago industry by

2010.

VISION

To be a leader in sago technology.

Role: To generate technology, know-how and expertise so as to ensure the industry will

continuously remain sustainable, efficient, competitive and resilient.

R & D THRUST AREAS

Crop improvement for high yield, good quality starch and maturity palms.

Mass propagation of high quality planting materials.

Understanding peat characteristics, reclamation and management technique for

optimum sago growth.

Agronomic and cultural practices.

Field mechanization and post-harvest handling.

Milling technology.

By-product utilization.

Product development.

Market studies and commercialization.

4

DIVISION

(A) UPSTREAM TECNOLOGY DIVISION (UTD)

The Upstream Technology Division undertake all aspects of upstream research of sago

palms which includes among others, breeding, propagation, agronomy, ecology and post-

harvest handling in order to develop modern and dynamic sago plantations.

Breeding and Propagation

Soil and Water Management

Agronomy

Crop Protection

Mechanization and Post-Harvest Technology

Objectives:-

To develop high yield, good quality starch and short maturity palms through

breeding and biotechnology.

To develop mass propagation of high quality planting materials.

To develop technology for peat soil, soil reclamation, utilization and

management.

To develop a water management system for sago plantation on peat.

To develop cost-effective nutrient management and cultural practices for sago on

peat.

To develop economical sago-based crop combinations.

To select appropriate machineries and develop a mechanization system that

minimizes drudgery and increase post-harvest handling efficiency for preservation

of log (starch) quality.

5

(B) DOWNSTREAM TECHNOLOGY DIVISION (DTD)

The Downstream Technology Division conducts commercially oriented scientific

research on sago in order to facilitate the establishment of high technology sago-based

industry in food and non-food areas in a form of support as either technological know-

how or product development or consultation services in related areas. This division will

cover strategic research areas including analytical studies, milling technology, by-product

utilization and product improvement and development.

Analytical Studies

Milling Technology

By-Product Utilization

Product Improvement and Development

Objectives:-

To develop competitive technology for refined sago starch production suitable for

downstream activities.

To value-add sago-based product in food and non-food areas.

To develop value added by-products via integrated palm utilization and zero

waste concept.

To generate consultancy services/ know-how for sago related enterprises.

(C) R & D COMMERCIALIZATION DIVISION

The R & D Commercialization Division is responsible for conducting studies on socio-

economics for sago industry. It is also responsible for managing and commercializing the

technologies generated internally or acquired externally, undertake the development of

business enterprise and provide commercial technical advisory and laboratory services

and training.

6

Objectives:-

To transfer and commercialize sago-based products and services generated from

CRSB for the generation of revenue.

To develop market network for the development of sago industry demand.

To coordinate advisory work and consultancy.

(D) CORPORATE SERVICES DIVISION

The Corporate Service Division serves in a supporting role to coordinate and monitor

research development plans, infrastructural plans, establishing and formulating a full

spectrum of financial and administrative functions. It also serves human resource

management development and enhancement of information technology system in tandem

with the company’s overall corporate and business plan.

Administration

Finance

Information centre

Human resource

Objectives:-

The Corporate Service Division is a central service division to support the

functions and research and development activities of the company.

7

CHAPTER 2: INDUSTRIAL TRAINING PROJECT

TITLE: EFFECTS OF ANTIBIOTICS ON ENDOGENOUS BACTERIAL

CONTAMINATION IN INITIATION STAGE OF SAGO PALM

INTRODUCTION

Plant tissue culture is a well-known technique used to propagate plants to a large

number of identical individuals. This technique is widely used by people in

commercialization of certain species of plants and research in laboratory throughout the

world. However, contamination usually occurred on the explant being cultured and this

becomes a known serious problem that was faced by most researchers.

Contamination on the explant might be come from environment, personnel, or

endophytic microorganisms. Endophytic or endogenous microorganisms such as bacteria,

fungi, yeast and others are microorganisms that can be found in the plant itself and not

from other sources. Normally, this kind of microorganism cannot be eliminated during

surface-sterilization of the plant sample in initiation stage of plant tissue culture and their

presence cannot be detected in early stage of culture. These problems can affect the result

of research and reduce the production of commercial plantlets.

This project is more emphasized on the endogenous bacterial contamination.

From the related research by other people, both type (Gram-positive and Gram-negative)

bacteria are found as endogenous bacteria in different plant species and there are various

methods were reported by other people that were effective in eliminating this endogenous

bacterial contamination such as by medium acidification (Leifert et. al., 1993), hot water

treatment (Ferrador et. al., n. d ), addition of formaldehyde (Nirmala et. al., 1992) and the

use of antibiotics (Tanprasert & Reed, 1997; Habiba et. al., 2002; Tal et. al., n. d.;

Kneifel & Leonhardt, 1991; Chanprame et. al., 1996).

For this project, we emphasized on the use of antibiotics due its simplicity and the

availability of resources. Antibiotics are substances that destroy or inhibit the growth of

8

microorganisms, particularly disease-producing bacteria and fungi. Antibiotics are

obtained from microorganisms (especially moulds) or synthesized. However, the overuse

of antibiotics can lead to the development of resistant strains of microorganisms (Oxford,

2004).

Endogenous bacterial contamination becomes our major problem in propagating

sago palm because it causes the waste of sample. This is because sometimes this kind of

contamination is latent and only visible when the plantlet transferred to the open area

(stage 4 of micropropagation). So, early prevention at early stage (initiation stage or

establishment stage) of sago palm tissue culture is needed to eliminate this problem

before it getting worse. In order to solve this problem, this project was planned and it is

more concerned to test the effects of the use of different antibiotics with various

concentrations on endogenous bacterial contamination in initiation stage of sago palm.

OBJECTIVES:

To determine which antibitotic that is most reactive on endogenous bacterial

contamination in initiation stage of sago palm.

To determine the minimum concentration of antibiotic that enough to eliminate

bacterial contamination.

9

MATERIALS AND METHODS

(A) BACTERIAL PREPARATION

Materials:-

2 bacteria sources: a) batch A b) batch B

Sterile distilled water

LB broth

Inoculating loop

Bunsen burner

10 plates of nutrient agar (NA)

4 plates of PDA

Parafilm tape

Micropippete

Permanent marker pen

Methods:-

1. Bacterial source (batch A) was taken aseptically using inoculating loop.

2. Then, the inoculating loop was dipped an stirred in a bottle containing 2 ml LB broth

to make a bacterial culture.

3. The bacterial culture was incubated overnight with shaker at 37 ºC.

4. After overnight, 200 µl of bacterial culture was pippeted and smeared on media. 5

plates NA medium and 2 plates PDA medium were used.

5. The bacterial culture was also streaked aseptically onto medium using inoculating

loop. 5 plates NA medium and 2 plates PDA medium were also used. This streaking

technique was used to produce a single colony of bacteria.

6. Above steps (1-5) was repeated using another bacterial source (batch B).

7. When the bacterial culture on media was dried, the petry dishes was covered with

parafilm.

10

040723L4FSDay 4c/5 (17/5)

040722 (a)CT1001Day 4c/5 (18/5)

8. All the samples was incubated overnight at 37 ºC in incubater. There were no growth

of bacterial colony.

9. The samples was stored in room temperature overnight and there were many bacterial

colony was grew.

10. The bacterial colony produced was stored at 4 ºC in the freezer before used.

(B) DISCS PREPARATION

Materials:-

Whatman filter paper

Hole-puncher

Autoclaved box

Forcep

Methods:-

1. Whatman filter papers were punched with hole-puncher to make a 0.5 diameter discs.

2. The discs was placed in a box and autoclaved at 121˚C for 20 minutes.

3. Aseptically, the discs will be dip into the sterilized antibiotic solvents at different

concentration.

4. Then, the antibiotic discs will be put at the centre of the Petri dish containing nutrient

agar and bacterial culture.

11

(C) ANTIBIOTICS STOCK PREPARATION ( 5 mg/ml )

Materials:-

6 types of antibiotics powder: (streptomycin sulphate, gentamicin sulphate,

penicillin G sodium, cefotaxime sodium,

carbenicillin disodium, and rifampicin)

Forceps

Spatula

Sterile beakers (50 ml)

Sterile filter and syringe



Aluminium foil

Eppendorf tube

Measuring cylinder

Methods:-

1. 0.05 g of each antibiotic powder was weighted.

2. Then, the antibiotic powder were dissolved with 10 ml sterile distilled water in 50 ml

beaker and stirred using spatula.

3. The beaker is covered with aluminium foil before it was filter sterilized in laminar flow

hood.

3. The antibiotic solution was filter sterilized twice and filled in the 1.5 ml eppendorf

tubes.

4. The eppendorf tubes were labeled and stored in freezer at -20 ˚C.

5. The antibiotics must be thaw first before used.

12

(D) ANTIBIOTICS DILUTION ( 50, 100 & 150 mg/L )

Materials:-

Antibiotics stock

Beakers (50 ml)

Sterile measuring cylinder



Aluminium foil

Methods:-

1. Each of 6 different antibiotics was diluted with 3 different concentrations

(50, 100 and 150 mg/l).

2. 10 ml of each antibiotics solution was prepared for every concentration in a beaker.

3. The volume of antibiotics stock needed for every concentration was calculated as

below:

(i) 50 mg/L

M1 = 5 mg/ml M2 = 50 mg/ L 0.05 mg/ml

V1 = x V2 = 10 ml

M1V1 = M2V2

5 (x) = 0.05 ( 10 )

5 x = 0.5

x = 0.1 ml

x = 100 µl

13

(ii) 100 mg/L

M1 = 5 mg/ml M2 = 100 mg/ L 0.1 mg/ml

V1 = x V2 = 10 ml

M1V1 = M2V2

5 (x) = 0.1 ( 10 )

5 x = 1

x = 0.2 ml

x = 200 µl

(iii) 150 mg/L

M1 = 5 mg/ml M2 = 150 mg/ L 0.15 mg/ml

V1 = x V2 = 10 ml

M1V1 = M2V2

5 (x) = 0.15 ( 10 )

5 x = 1.5

x = 0.3 ml

x = 300 µl

4. Each volume of antibiotics calculated above was top up with sterile distilled water to

get 10 ml final volume of antibiotics solution.

5. The beakers were covered with aluminium foil.

14

E) ANTIBIOTICS CLEAR ZONE TEST

Materials:-

Diluted antibiotics (50, 100 &150 mg/L)

Isolated bacterial culture

144 fresh NA media

Forceps

Sterile bijou bottles

Micropippete

Spreader

Whatman filter paper discs



Aluminium foil

Parafilm

Ruler

Methods:-

1. 2 colonies from isolated bacterial culture were taken aseptically using toothpicks.

2. The bacterial colonies were dissolved in 3 ml sterile distilled water in a bijou bottle.

3. 200 µl of bacterial culture was pippeted and smeared on the 144 fresh nutrient agar

(NA).

4. The bacterial culture on the media was spreaded using sterile spreader until dried.

5. The filter paper discs were dip in each diluted antibiotics solution.

6. Then, the antibiotic discs was put at the centre of the Petri dish containing nutrient

agar and bacterial culture.

7. All the Petri dishes was covered using parafilm an incubated in room temperature.

8. Result is collected by measuring the diameter of clear zone that will be produced

around the antibiotic discs.

9. All the data was recorded in the table.

15

PROJECT SCHEDULE

WEEK /

ACTIVITIES

1 2 3 4 5 6 7 8

Project

proposal

preparation

Bacterial

isolation

Antibiotic

preparation

Nutrient agar

medium

preparation

Antibiotic clear

zone test

Analysis of

results

Report writing

Presentation

16

GRAPH OF CLEAR ZONE DIAMETER VS. DAY

0

0.5

1

1.5

2

2.5

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

day

clea

r zo

ne

dia

me

ter

strep50

strep100

strep150

genta50

genta100

genta150

penG50

penG100

penG150

carbe50

carbe100

carbe150

cefo50

cefo100

cefo150

rif50

rif100

rif150

RESULT

Source A

Concentration (mg/L) 50 100 150

Antibiotic Day 1 2 3 4 7 14 1 2 3 4 7 14 1 2 3 4 7 14streptomycin sulphate 2.37 2.37 2.37 2.33 2.30 2.30 2.57 2.53 2.53 2.50 2.50 2.47 2.56 2.67 2.67 2.63 2.60 2.57gentamicin sulphate 1.77 1.87 1.65 1.55 1.55 1.50 1.93 2.07 2.07 2.07 2.07 2.07 1.90 1.93 1.97 1.93 1.90 1.90penicillin G sodium 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00carbenicillin disodium 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.63 0.67 0.00 0.00 0.00 0.00 0.90 1.00cefotaxime 0.00 0.70 0.70 0.70 0.70 0.70 1.25 1.30 1.25 1.15 1.15 1.10 1.15 1.20 1.20 1.20 1.20 1.10rifampicin 0.00 0.70 0.70 0.80 0.65 0.70 0.00 0.70 0.70 0.73 0.70 0.70 0.87 0.93 0.97 0.93 0.93 0.90

17

GRAPH OF CLEAR ZONE DIAMETER VS. DAY

0

0.5

1

1.5

2

2.5

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

day

clea

r zo

ne

dia

me

ter

(cm

)

strep50

strep100

strep150

genta50

genta100

genta150

penG50

penG100

penG150

carbe50

carbe100

carbe150

cefo50

cefo100

cefo150

rif50

rif100

rif150

Source B

Concentration (mg/L) 50 100 150 Antibiotic Day 1 2 3 4 7 14 1 2 3 4 7 14 1 2 3 4 7 14streptomycin sulphate 2.03 2.10 2.10 2.10 2.07 2.17 2.23 2.23 2.23 2.20 2.20 2.23 2.67 2.73 2.70 2.70 2.70 2.50gentamicin sulphate 1.87 1.90 1.90 1.87 1.83 1.83 1.80 2.20 1.93 1.83 1.80 1.80 2.13 2.20 2.17 2.10 2.03 2.00penicillin G sodium 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.00 1.00 0.80 0.80carbenicillin disodium 0.00 0.00 0.00 0.00 0.00 0.70 0.00 0.00 0.00 0.00 0.67 0.70 0.00 0.00 0.00 0.00 0.73 0.80cefotaxime 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.83 0.83 0.80 0.80 1.73 1.77 1.73 1.70 1.63 1.60rifampicin 0.00 0.00 0.70 0.70 0.70 0.70 0.00 0.00 0.70 0.70 0.67 0.70 1.25 1.07 1.07 1.10 1.10 1.05

18

DISCUSSIONS

The main source of our bacteria samples were from 2 different contaminated

culture medium of explants; Source A-040723 L4FS at day 4(without charcoal) and

Source B-040722a CT1001 at day 4 (with charcoal). After isolation step, we assumed

that the endogenous bacteria from Source A and B are from the same genus since the

colonies produced by both source are morphologically same. Besides that, there was no

colony observed on plates incubated at 37˚C for overnight. The colonies only appear

when the plates were incubated at room temperature (25˚C). We made assumption based

on this reason that the bacteria used in this project were not come from human being.

In this project, there were 6 different types of antibiotics being used because of

their availability in the laboratory. They are streptomycin sulphate, gentamicin sulphate,

penicillin G sodium, cefotaxime, carbenicillin disodium and rifampicin. All of them are

prepared in stock condition since antibiotic can easily degraded and very sensitive to

light. Due to this two factors, antibiotics solution were sterilized by using filter-

sterilization method and the dilution of these antibiotics to different concentration was

only done when the clear zone test is ready to be performed.

All these 6 antibiotics have ability to react towards different kind of bacteria;

either Gram positive or Gram negative bacteria respectively (refer to the table 1 below)

No. Antibiotics Type of bacteria that sensitive to it

1 Streptomycin sulphate Gram negative (-ve)

2 Gentamicin sulphate Gram negative (-ve)

3 Penicillin G. sodium Gram positive (+ve)

4 Carbenicillin disodium Gram negative (-ve)

5 Cefotaxime Gram negative (-ve)

6 Rifampicin Gram negative (-ve) and Gram positive (+ve)

Table 1: 6 type of antibiotics with type of bacteria that sensitive to them respectively

Based on the above table (Table 1), we found that the endogenous bacteria that we used

in this project is Gram negative (-ve) bacteria since only plates treated with penicillin G

sodium did not shown any clear zone.

19

According to our result, 5 antibiotics except penicillin G sodium react

immediately towards the bacteria since we can see the clear zone formed around the

antibiotic discs on the next day after the bacteria was spread onto the nutrient agar and

the antibiotic disc was placed in the middle of the plates. However, their activities were

decreased through the day and can last for 2 weeks (14 days). The best 4 antibiotics that

we found can be used in order to eliminate the endogenous bacteria are streptomycin

sulphate, gentamicin sulphate, cefotaxime and rifampicin and all of these were effective

with 150 mg/L concentration. However, the result show gentamicin and cefotaxime treat

to source A is most effective at 100 mg/L concentration. This weird result might be due

to personnel error during pipetting the antibiotic stock solution to make dilution of it.

Although we had worked with very sterile condition, contamination was still

occurred. There are several plates contaminated by insect larvae. This contamination

might be due to the use of parafilm to seal the Petri dish. Since we incubate all the plates

at room temperature and the plates were exposed to the air-conditioner, the parafilm

become exhausted and allowed the insects to lay their eggs into the plates that contain

much nutrient for the development of their eggs.

CONCLUSION

The most effective antibiotic against endogenous bacteria of sago palm

streptomycin sulphate with 150 mg/L concentration.

The effectiveness of antibiotics can lasts for 2 weeks even though it decreases

through the days.

20

CHAPTER 3: ADDITIONAL WORK

1. LITERATURE REVIEW

Cost Reduction via Alternative Gelling Agent in Plant Tissue Culture

Activities done:

Searching information (articles, journals, etc. ) related to the title.

Doing summary of information in table form.

2. MEDIUM PREPARATION ROOM WORK

Activities done:

Covering flasks containing medium with aluminium foil and then tied with rubber

band before autoclaving them.

Washing flasks containing wrong-made medium.

Helping in filter-sterilization of AR sucrose.

Cutting aluminium foil into smaller pieces according to flasks sizes.

Weighing certain ingredients for certain medium.

3. OTHER WORKS

Activities done:

Helping in labeling and parafilm the flasks containing the subcultured explants.

21

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