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Transcript of 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
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.
12
(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 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
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 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