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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 CHAPTER 1: CRAUN RESEARCH SDN. BHD. 1. Introduction 2. Mission 3. Vision 4. R & D Thrust Areas 5. Division (a) Upstream Technology Division (b) Downstream Technology Division (c) R & D Commercialization Division (d) Corporate Services Division CHAPTER 2: INDUSTRIAL TRAINING PROJECT 1. Introduction 2. Materials and Methods 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

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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. CRAUNs 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.

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.

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 postharvest 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.

(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 knowhow 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 socioeconomics 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 companys 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.

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 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.

MATERIALS AND METHODS (A) BACTERIAL PREPARATION Materials: 2 bacteria sources: a) batch A 040723 L4FS Day 4 c/5 (17/5) 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 b) batch B 040722 (a) CT1001 Day 4 c/5 (18/5)

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 C. 4. After overnight, 200 l 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.

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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. 8. All the samples was incubated overnight at 37 C 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 C 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 121C 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.

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(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 Sterile distilled water Permanent marker pen 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.

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(D) ANTIBIOTICS DILUTION ( 50, 100 & 150 mg/L ) Materials: Antibiotics stock Beakers (50 ml) Sterile measuring cylinder Sterile distilled water Permanent marker pen 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 V1 = x M1V1 = M2V2 5 (x) = 0.05 ( 10 ) 5 x = 0.5 x = 0.1 ml x = 100 l M2 = 50 mg/ L V2 = 10 ml 0.05 mg/ml

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(ii) 100 mg/L M1 = 5 mg/ml V1 = x M1V1 = M2V2 5 (x) = 0.1 ( 10 ) 5x =1 x = 0.2 ml x = 200 l M2 = 100 mg/ L V2 = 10 ml 0.1 mg/ml

(iii) 150 mg/L M1 = 5 mg/ml V1 = x M1V1 = M2V2 5 (x) = 0.15 ( 10 ) 5 x = 1.5 x = 0.3 ml x = 300 l 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. M2 = 150 mg/ L V2 = 10 ml 0.15 mg/ml

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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 Sterile distilled water Permanent marker pen 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 l 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.

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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.

PROJECT SCHEDULE WEEK / ACTIVITIES Project proposal preparation Bacterial isolation Antibiotic preparation Nutrient agar medium preparation Antibiotic clear zone test Analysis of 1 2 3 4 5 6 7 8

results Report writing Presentation

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RESULT Source A
Concentration (mg/L) 50 Antibiotic
Day

100

150

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14

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streptomyci n sulphate gentamicin sulphate penicillin G sodium carbenicillin disodium cefotaxime rifampicin

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 2.57 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 1.90 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 0.00 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 1.00 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 1.10 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

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Source B
Concentration (mg/L) 50 100 7 14 1 2 3 4 7 14 1 2 3 150 4 7 14

Antibiotic
Day

streptomyci n sulphate gentamicin sulphate penicillin G sodium carbenicillin disodium cefotaxime rifampicin

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 2.50 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 2.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.00 1.00 0.80 0.80 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 0.80 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 1.60 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

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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 37C for overnight. The colonies only appear when the plates were incubated at room temperature (25C). 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 filtersterilization 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

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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. 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.

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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.

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