All the steps in this experiment were performed under aseptic conditions. Good sterilization techniques are the first and most important step in ensuring consistent results throughout the whole project. All apparatus, culture media and distilled water in the experiment was autoclaved at 121ËšC for 20 minutes to sterilize everything and to avoid contamination. The experiment was conducted in the Laminar Flow hood, and since contaminating bacteria are ubiquitous and are found on fingertips, bench tops, and almost everywhere in the environment, it is important to minimize contact with these contaminating surfaces and disinfect all surfaces prior to and after use.
3.2 Preparation of growth media for bacterial culture
3.2.1 Preparation of Okon mediua
For rapid multiplication of rhizobacteria, it was grown in a liquid media with added nitrogen source was chosen. In such media, with rapid stirring or shaking, cell concentration of 108 can be reached by 24-72 hours. To stabilize the pH at a desired value upon prolonged growth, buffer was added.
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In Okon media preparation, each part of the medium was prepared individually first before it was mixed together. Buffer was prepared separately and added upon bacteria inoculation to the media. All components were mixed with less than 1 L of distilled water in a beaker and stirred with a magnetic stirrer. The pH was then adjusted to 6.8. When the pH stabilized, then water was added up to 1L using a volumetric flask. Following this, 100 ml of the solution was poured into each of 10 conical flasks. The conical flask neck was then covered with cotton and wrapped with aluminum foil prior to autoclaving at 121 ËšC for 20 minutes. (Okon et al,1977). This Okon media was used for all bacteria culture and also for all bacteria inoculation.
3.2.2 Preparation of Congo Red (RC) agar
Congo red is an isolation medium, whereby it is selective for distinguishing between rhizobacteria and other bacteria. It is a nitrogen free media whereby bacteria that do not fix nitrogen will not be able to live on it. All chemicals needed were added to a beaker containing water less than 1L. The pH was then adjusted to 7.0 with 0.1 N KOH and distilled water was added to make up a final volume of 1L. The medium was autoclaved at 121Â°C for 20 min. In addition, 15 ml of a 1:400 aqueous solution of Congo red was autoclaved separately and added aseptically to each liter of the melted medium just before agar plates were prepared. RC agar was used in this experiment for streaking method and also c.f.u (viable cell count) evaluation.
3.2.3 Preparation of nitrogen free semisolid malate medium (Nfb medium)
Nfb medium is prepared prior to Acetylene reduction assay for free-living bacteria. All isolates was incubated in Nfb semisolid media for 72 hours for pellicle formation. 5% acetylene was injected into the universal bottle and incubated for 24 hours at 30ËšC. After incubation, 1 ml of gas was withdrawn and injected to the gas chromatography for Acetylene Reduction Assay.
3.3 Preparation of bacterial culture and bacteria stock.
Bacterial culture was prepared for this experiment from the isolate obtained from the lab. Six treatments involved in this experiment were the inculation with L2, L15, SP7, Z78 plus two controls which included a negative control treatment (SP7K) and a positive control (+100N). A loopful of colony was taken and inoculated to Okon media and then put on a shaker at 160 rpm for 24 to 72 hours. After 48 hours, the culture was streaked on RC plate to check the purity of the bacterial culture. If no contamination was detected, the bacterial culture was transferred to glycerol stock and stored in freezer at -21ËšC to ensure longer storage life. OD readings was taken before the transfer of bacteria culture to glycerol stock.
Table 3.3: Type of treatment administered
Negative control (SP7K)
Azospirillum sp. (kill)
Positive Control (+N)
100% N fertilization
3.4 Absorbance reading (spectrophotometer)
Absorbance reading is done to measure turbidity and by determining the amount of light scattered by a suspension of cells.Â Particles such as bacteria will scatter light in proportion to their numbers. The turbidity or optical density (OD) of a suspension of cells is directly related to cell mass or cell number. The higher the reading of the spectrophotometer, the higher will be the number of cell in the culture will be. The method is simple and nondestructive, but the sensitivity is limited to about 107 cells per ml for most bacteria.
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2ml from the culture stock was transferred to a cuvette. Okon medium that was used to prepare the bacterial culture was used to calibrate the spectrophotometer to zero. OD reading was taken at a wavelength of 600nm. The culture in the cuvette was diluted if the OD reading is more than 1.0. The reading from the sample culture was repeated three times to avoid errors in reading. Throughout the experiment, OD reading was taken every time the bacteria culture was prepared and preceded to another step. This is to obtain information on how concentrated the culture is and also to standardize the size of inoculums during bacteria inoculation.
3.5 Viable cell count
Viable cell counts, also called plate counts, involve plating out a sample of a culture on a nutrient agar surface. For this experiment, drop plate count method was used. The sample or cell suspension was diluted in sterile distilled water before plating.Â For each sample the dilution was prepared from 10-1 to 10-8. For each strain and for each dilution in the series, fresh pipette tips were used. Then, 50 Âµl of the bacterial culture was dropped on a section of the plate. Each dilution had 3 replicates of same dilution to have a better result. The plate were incubated and observed after 48 hours. Each viable unit grows and forms a colony. Each colony which can be counted is called a colony forming unit (cfu) and the number of cfu is related to the viable number of bacteria in the sample. Only colonies between the range of 30 to 300 are considered and counted. The formula to obtain the number of cells per ml of the bacterial culture is:
(number of colonies) X (dilution factor) X (vol. of inoculum) = Ï‡ cell per ml
3.6 Indole Acetic Acid Assay (IAA)
An estimation of IAA production by diazotrophs was assayed using spectrophotometric analysis. The tested bacteria were cultured in 250 mL conical flask containing 100 mL Okon media supplemented with 0.5 g L-1 L-tryptophan (as a physiological precursor for biosynthesis of IAA in plants and microbes) (Glickmann and Dessaux, 1995). The bacterial cultures were shaken at 160 rpm, at room temperature (30-32ï‚°C). After 2 days, the bacterial cultures were harvested and the bacterial cultures were transferred to eppendorf tube aseptically and centrifuged under 5000 rpm for 15 minutes. Then, 2 ml of supernatant and 3 ml of Salkowski reagent were vortexed then incubated in the dark for 30 minutes. Changes of colour will occur and measured by spectrophotometer (530 nm). The concentration of IAA in each culture medium was determined by comparison with a standard curve
3.7 Acetylene Reduction Assay (ARA)
ARA was a test to check the ability of the isolate to fix nitrogen. In this whole experiment, ARA test was conducted twice. The first was to test the nitrogen fixing capabilities of the isolate alone in the free-living condition. Another ARA test was conducted towards the end of the experiment after the paddy plant was harvested and the roots of the plants were tested to observe if the isolate colonizing its roots was able to fix nitrogen under associated conditions.
For free-living ARA, bacterial cultures were grown on 10 ml of semi-solid NFb medium in 30 ml universal bottles sealed tightly with the cap containing rubber septa. The bottle then incubated for 72 hours for pellicle formation. 72 hours after the inoculation, a 5% volume, 1ml of gas was sucked out and replaced with 5% volume, 1ml of acetylene gas (Burris, 1972). The amount of ethylene was measured after 24 hours of incubation with the actylene. All incubations were done at room temperature in the dark, avoiding any movement of the vials. Ethylene was measured using a Shimadzu Gas Chromatograph (GC-2014). Calculations were based on peak area. To determine the cell number, serial dilutions of the culture were performed at the end of the incubation, plated on RC agar and then counted after incubation at 30 Â°C for 3 to 5 days.
For associated bacteria ARA, the whole roots of each treatment was taken after harvest and incubated in an airtight vessel (1050 mL) containing 600 mL of sterile distilled water for 6 hours. After 6 hours, water was drained and gas in the airtight vessel was sucked out and replaced with nitrogen gas N2:O2 (70:30) and incubated overnight. Next, nitrogen gas was removed and replaced with 945 mL of Ar:O2 (80:20) (90% volume of the vessels) and 105 mL of pure acetylene (10% volume of the vessels) (Abrantes et al., 1976: Baldani and Baldani, 2005). After 6 hours of incubation, 1 ml of gas was taken in 8 replicate for each treatment and proceed with ARA using Shimadzu Gas Chromatograph (GC-2014).
3.8 Paddy Planting
3.8.1 Seed surface sterilization and seed germination
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Healthy seeds were selected to optimize the chances for germination. Seeds prepared were soaked in 70% of alcohol and shaken vigorously for 10 minutes. Then the seeds were washed with sterile distilled water for 3 times. The distilled water was then drained off and a house detergent (NaOCl) with the pH 7.0 was added and shaken vigorously for 8 minutes. Then the paddy seeds were transferred to a conical flask containing sterile distilled water. The paddy seeds were washed with sterile distilled water for 3 times and at the third time the seeds which were soaked in sterile distilled water were put on a shaker for 30 minutes. After 30 minutes, without draining the contents of the conical flask, it was incubated in an oven at 60ËšC for 2 hours. Next, after incubation, the seeds were rinsed with sterile distilled water for the final time and prepared for the germination step.
For seed germination, autoclaved Petri dish with cotton inside was soaked with sterile distilled water. Then seed were placed on top of the wet cotton, wherein for each petri dish 5 seeds were placed. Seeds were then germinated in a closed and dark environment. The germination period is around 3 to 5 days.
3.8.2 Soil preparation
For paddy planting, aerobic condition was implemented instead of flooded condition. The soil was sieved before use to separate foreign materials from the soil. The ratio of soil and sand used was 3:1. For each polythene bag, 1.5 kg of mixture of soil and sand mixture were used. For every treatment in the experiment, 5 replicates were prepared. 30 polythene bags were prepared and the soil was ensured to be moist before the paddy plantation.
3.8.3 Bacteria inoculation
Throughout the whole experiment, bacteria inoculation was administered three times. The first 2 inoculations were done during the planting phase. To prepare the bacteria culture, the bacteria stock was added to Okon media. After purity check, cfu, standardization of inoculums by comparison to McFarland and OD readings were taken, the bacteria culture is ready to be inoculated.
First, when all of the seeds have germinated, the seeds were soaked in the bacteria culture. All germinated seeds were soaked in the bacteria culture according to the treatment allocated. The seeds were incubated with the bacteria culture for 1 hour (first inoculation). Then, after incubation, the seeds were transferred aseptically to a sterile petri dish according to the treatment administered and preceded with planting.
For the second inoculation of bacteria, after the paddy was planted, 50 ml of bacteria was added to the newly planted seed. This is called a liquid inoculation. Last inoculation (liquid inoculation) of bacteria was done during week 5 to reinforce the bacterial colonization on the plant.
3.8.4 Planting method
During planting, for each polythene bag, 3 seeds were planted to ensure a higher survival rate of the replicates. On the second week, only 1 plant was allowed to grow in each polythene bag to avoid competition for light, water, and nutrient. The plants were watered twice daily.
3.9 Fertilizer administration to the plant.
Fertilizer was given to the plant to ensure better growth of the plants and higher survival rates.
Table 3.9: Amount of fertilizer given to the paddy plant.
g/kg (in 1.5 kg soil)
2.15 X 10-2
2.84 X 10-2
2.28 X 10-4
5.38 X 10-3
7.10 X 10-3
5.69 X 10-5
2.37 X 10-2
2.13 X 10-4
9.45 X 10-3
2.45 X 10-4
3.10 Non-destructive sampling
Non-destructive sampling was conducted throughout the experiment to monitor the plant growth. Sampling was conducted once every week until harvest which was on the 10th week. So a total of 10 observations were made.
3.10.1 Plant height
Plant height was measured using a measuring tape. The unit of measurement used was in centimeter.
3.10.2 Total number of leaves
The number of leaves was recorded for each plant according to its treatment.
3.10.3 Chlorophyll contents (standard curve??)
Chlorophyll content was measured using SPAD meter (Minolta SPAD Meter 502). Three different levels of leaf greenness were measured with the SPAD meter. The values measured must be within a certain range. Dark green is in the range of 40-50, light green is between 20-40 and yellow is below 20. For every leaf, the chlorophyll content was measured in 3 different spot. The average of the readings was taken and recorded.
3.11 Harvesting paddy plant.
Paddy plants were harvested after 70 days. Before the final harvest, non-destructive sampling was conducted. Each polythene bag was cut at its side to harvest the paddy plant. The paddy plant was then separated between the leaves and its roots.
The roots with soil were placed under running water to wash away the soil. The length of the roots were first measured and then weighed to get the fresh weight. The whole root for each treatment was removed to undergo ARA test. The rest of the roots were incubated in the oven at temperature of 60ËšC for 4 days.
For the leaves, after the fresh weight was taken,they were put inside a petri dish according to their treatment. They were then incubated in an oven with temperature of 60ËšC.
Dry weight of both parts (leaves and root) of the paddy plant was taken over several days. This was done by taking out the roots and leaves from the oven and weighing them. The dry weight is obtained when the weight reading became constant.
3.12 Determination of Protein Content
Leaves of each replicate for every treatment were grinded and filtered to get the leaf extract. 0.1 ml of leaf extract were placed into test tubes and left for 10 minutes at room temperature. Then, 2 ml of D solution was added into the test tubes and left for another 10 minutes. Finally 0.2 ml of folin-phenol solution was added and left for 30 minutes before OD reading at 600 nm was taken using spectrophotometer.
The determination of standard curve of protein uses the reagent 1mg/ml Bovine Serum Albumine: 5mg in 5ml water (1Âµ/Âµl).
2% Na2CO3 in 0.1 N NaOH
1% NaK Tartarate in dH2O
0.5% CuSO4.5H2O in dH2O
48ml of A + 1ml of B + 1ml of C
Phenol reagent; 1 folin-phenol:1 distilled water
3.13 Statistical Analysis
Appendix stuff frm m&m
Table 3.2.1: Chemicals for Okon medium
Table 3.2.2: Chemicals for RC agar
Table 3.2.3: Chemicals for Nfb semisolid Malate media
*BTB (Bromo Thymol Blue)
g/L (0.2N KOH)