Rapid Multiplication Of Rhizobacteria From Okon Meduia Biology Essay

All the stairss in this experiment were performed under sterile conditions.

Good sterilisation techniques are the first and most of import measure in guaranting consistent consequences throughout the whole undertaking. All setup, civilization media and distilled H2O in the experiment was autoclaved at 121EsC for 20 proceedingss to sterilise everything and to avoid taint. The experiment was conducted in the Laminar Flow goon, and since polluting bacteriums are omnipresent and are found on fingertips, bench tops, and about everyplace in the environment, it is of import to minimise contact with these polluting surfaces and disinfect all surfaces prior to and after usage.

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3.2 Preparation of growing media for bacterial civilization

3.2.1 Preparation of Okon mediua

For rapid generation of rhizobacteria, it was grown in a liquid media with added N beginning was chosen. In such media, with rapid stirring or agitating, cell concentration of 108 can be reached by 24-72 hours.

To stabilise the pH at a coveted value upon drawn-out growing, buffer was added.In Okon media readying, each portion of the medium was prepared separately first before it was assorted together. Buffer was prepared individually and added upon bacteriums vaccination to the media.

All constituents were assorted with less than 1 L of distilled H2O in a beaker and stirred with a magnetic scaremonger. The pH was so adjusted to 6.8.

When the pH stabilized, so H2O was added up to 1L utilizing a volumetric flask. Following this, 100 milliliter of the solution was poured into each of 10 conelike flasks. The conelike flask cervix was so covered with cotton and wrapped with aluminium foil prior to autoclaving at 121 EsC for 20 proceedingss. ( Okon et al,1977 ) .

This Okon media was used for all bacteriums civilization and besides for all bacteriums vaccination.

3.2.2 Preparation of Congo Red ( RC ) agar

Congo red is an isolation medium, whereby it is selective for separating between rhizobacteria and other bacteriums. It is a nitrogen free media whereby bacteriums that do non repair N will non be able to populate on it. All chemicals needed were added to a beaker incorporating H2O less than 1L. The pH was so adjusted to 7.0 with 0.

1 N KOH and distilled H2O was added to do up a concluding volume of 1L. The medium was autoclaved at 121A°C for 20 min. In add-on, 15 milliliter of a 1:400 aqueous solution of Congo red was autoclaved individually and added aseptically to each litre of the liquid medium merely earlier agar home bases were prepared. RC agar was used in this experiment for streaking method and besides c.f.u ( feasible cell count ) rating.


3 Preparation of nitrogen free semisolid malate medium ( Nfb medium )

Nfb medium is prepared prior to Acetylene decrease check for nonparasitic bacteriums. All isolates was incubated in Nfb semisolid media for 72 hours for pellicle formation. 5 % ethyne was injected into the cosmopolitan bottle and incubated for 24 hours at 30EsC. After incubation, 1 milliliter of gas was withdrawn and injected to the gas chromatography for Acetylene Reduction Assay.

3.3 Preparation of bacterial civilization and bacterium stock.

Bacterial civilization was prepared for this experiment from the isolate obtained from the lab. Six interventions involved in this experiment were the inculation with L2, L15, SP7, Z78 plus two controls which included a negative control intervention ( SP7K ) and a positive control ( +100N ) .

A loopful of settlement was taken and inoculated to Okon media and so set on a shaker at 160 revolutions per minute for 24 to 72 hours. After 48 hours, the civilization was streaked on RC home base to look into the pureness of the bacterial civilization. If no taint was detected, the bacterial civilization was transferred to glycerol stock and stored in deep-freeze at -21EsC to guarantee longer storage life. OD readings was taken before the transportation of bacteriums civilization to glycerol stock.Table 3.3: Type of intervention administeredTreatmentStrainL2Enterobacter asburiaeL15Gluconacetobacter DiazotrophicusSP7Azospirillum sp.

Z78Herbaspirillum sp.Negative control ( SP7K )Azospirillum sp. ( putting to death )Positive Control ( +N )100 % N fertilisation

3.4 Absorbance reading ( spectrophotometer )

Optical density reading is done to mensurate turbidness and by finding the sum of visible radiation scattered by a suspension of cells.

A Atoms such as bacteriums will disperse visible radiation in proportion to their Numberss. The turbidness or optical denseness ( OD ) of a suspension of cells is straight related to cell mass or cell figure. The higher the reading of the spectrophotometer, the higher will be the figure of cell in the civilization will be.

The method is simple and nondestructive, but the sensitiveness is limited to about 107 cells per milliliter for most bacteriums.2ml from the civilization stock was transferred to a cuvette. Okon medium that was used to fix the bacterial civilization was used to graduate the spectrophotometer to zero. OD reading was taken at a wavelength of 600nm. The civilization in the cuvette was diluted if the OD reading is more than 1.0. The reading from the sample civilization was repeated three times to avoid mistakes in reading. Throughout the experiment, OD reading was taken every clip the bacteriums civilization was prepared and preceded to another measure.

This is to obtain information on how concentrated the civilization is and besides to standardise the size of inoculants during bacteriums vaccination.

3.5 Feasible cell count

Feasible cell counts, besides called home base counts, involve plating out a sample of a civilization on a alimentary agar surface. For this experiment, bead home base count method was used. The sample or cell suspension was diluted in sterile distilled H2O before plating.A 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 Aµl of the bacterial civilization was dropped on a subdivision of the home base. Each dilution had 3 replicates of same dilution to hold a better consequence. The home base were incubated and observed after 48 hours. Each feasible unit grows and forms a settlement. Each settlement which can be counted is called a settlement organizing unit ( cfu ) and the figure of cfu is related to the feasible figure of bacteriums in the sample. Lone settlements between the scope of 30 to 300 are considered and counted.

The expression to obtain the figure of cells per milliliter of the bacterial civilization is:( figure of settlements ) X ( dilution factor ) Ten ( vol. of inoculant ) = I‡ cell per milliliter

3.6 Indole Acetic Acid Assay ( IAA )

An appraisal of IAA production by diazotrophs was assayed utilizing spectrophotometric analysis. The tried bacteriums were cultured in 250 mL conelike flask incorporating 100 milliliter Okon media supplemented with 0.

5 g L-1 L-tryptophan ( as a physiological precursor for biogenesis of IAA in workss and bugs ) ( Glickmann and Dessaux, 1995 ) . The bacterial civilizations were shaken at 160 revolutions per minute, at room temperature ( 30-32i‚°C ) . After 2 yearss, the bacterial civilizations were harvested and the bacterial civilizations were transferred to eppendorf tubing aseptically and centrifuged under 5000 revolutions per minute for 15 proceedingss. Then, 2 milliliter of supernatant and 3 milliliter of Salkowski reagent were vortexed so incubated in the dark for 30 proceedingss.

Changes of coloring material will happen and measured by spectrophotometer ( 530 nanometer ) . The concentration of IAA in each civilization medium was determined by comparing with a standard curve

3.7 Acetylene Reduction Assay ( ARA )

ARA was a trial to look into the ability of the isolate to repair N.

In this whole experiment, ARA trial was conducted twice. The first was to prove the N repairing capablenesss of the isolate entirely in the nonparasitic status. Another ARA trial was conducted towards the terminal of the experiment after the Paddy works was harvested and the roots of the workss were tested to detect if the isolate colonising its roots was able to repair N under associated conditions.For nonparasitic ARA, bacterial civilizations were grown on 10 milliliter of semi-solid NFb medium in 30 milliliter cosmopolitan bottles sealed tightly with the cap incorporating gum elastic septa. The bottle so incubated for 72 hours for pellicle formation. 72 hours after the vaccination, a 5 % volume, 1ml of gas was sucked out and replaced with 5 % volume, 1ml of ethyne gas ( Burris, 1972 ) .

The sum of ethene was measured after 24 hours of incubation with the actylene. All incubations were done at room temperature in the dark, avoiding any motion of the phials. Ethylene was measured utilizing a Shimadzu Gas Chromatograph ( GC-2014 ) . Calculations were based on peak country. To find the cell figure, consecutive dilutions of the civilization were performed at the terminal of the incubation, plated on RC agar and so counted after incubation at 30 A°C for 3 to 5 yearss.For associated bacterium ARA, the whole roots of each intervention was taken after crop and incubated in an air-tight vas ( 1050 milliliter ) incorporating 600 milliliter of sterile distilled H2O for 6 hours. After 6 hours, H2O was drained and gas in the air-tight vas was sucked out and replaced with N gas N2: O2 ( 70:30 ) and incubated overnight. Following, nitrogen gas was removed and replaced with 945 milliliters of Ar: O2 ( 80:20 ) ( 90 % volume of the vass ) and 105 milliliter of pure ethyne ( 10 % volume of the vass ) ( Abrantes et al.

, 1976: Baldani and Baldani, 2005 ) . After 6 hours of incubation, 1 milliliter of gas was taken in 8 replicate for each intervention and continue with ARA utilizing Shimadzu Gas Chromatograph ( GC-2014 ) .

3.8 Paddy Planting


8.1 Seed surface sterilisation and seed sprouting

Healthy seeds were selected to optimise the opportunities for sprouting. Seeds prepared were soaked in 70 % of intoxicant and agitate smartly for 10 proceedingss. Then the seeds were washed with unfertile distilled H2O for 3 times.

The distilled H2O was so drained off and a house detergent ( NaOCl ) with the pH 7.0 was added and shaken smartly for 8 proceedingss. Then the Paddy seeds were transferred to a conelike flask incorporating unfertile distilled H2O. The paddy seeds were washed with unfertile distilled H2O for 3 times and at the 3rd clip the seeds which were soaked in sterile distilled H2O were put on a shaker for 30 proceedingss. After 30 proceedingss, without run outing the contents of the conelike flask, it was incubated in an oven at 60EsC for 2 hours. Next, after incubation, the seeds were rinsed with unfertile distilled H2O for the concluding clip and prepared for the sprouting measure.

For seed sprouting, autoclaved Petri dish with cotton interior was soaked with unfertile distilled H2O. Then seed were placed on top of the wet cotton, wherein for each petri dish 5 seeds were placed. Seeds were so germinated in a closed and dark environment. The sprouting period is about 3 to 5 yearss.

3.8.2 Soil readying

For paddy planting, aerophilic status was implemented alternatively of afloat status. The dirt was sieved earlier usage to divide foreign stuffs from the dirt. The ratio of dirt and sand used was 3:1.

For each polyethylene bag, 1.5 kilogram of mixture of dirt and sand mixture were used. For every intervention in the experiment, 5 replicates were prepared.

30 polyethylene bags were prepared and the dirt was ensured to be damp before the paddy plantation.

3.8.3 Bacteria vaccination

Throughout the whole experiment, bacteriums vaccination was administered three times. The first 2 vaccinations were done during the planting stage. To fix the bacteria civilization, the bacteriums stock was added to Okon media. After pureness cheque, cfu, standardisation of inoculants by comparing to McFarland and OD readings were taken, the bacteria civilization is ready to be inoculated.First, when all of the seeds have germinated, the seeds were soaked in the bacteria civilization.

All germinated seeds were soaked in the bacterium civilization harmonizing to the intervention allocated. The seeds were incubated with the bacteriums civilization for 1 hr ( first vaccination ) . Then, after incubation, the seeds were transferred aseptically to a unfertile petri dish harmonizing to the intervention administered and preceded with planting.For the 2nd vaccination of bacteriums, after the Paddy was planted, 50 milliliter of bacterium was added to the freshly planted seed. This is called a liquid vaccination. Last vaccination ( liquid vaccination ) of bacterium was done during hebdomad 5 to reenforce the bacterial colonisation on the works.


8.4 Planting method

During planting, for each polyethylene bag, 3 seeds were planted to guarantee a higher survival rate of the replicates. On the 2nd hebdomad, merely 1 works was allowed to turn in each polyethylene bag to avoid competition for visible radiation, H2O, and food. The workss were watered twice daily.


9 Fertilizer disposal to the works.

Fertilizer was given to the works to guarantee better growing of the workss and higher endurance rates.Table 3.9: Sum of fertiliser given to the Paddy works.



g/kg ( in 1.

5 kilograms dirt )


100 % N( carbamide )142.15 X 10-2+N352.84 X 10-2562.28 X 10-425 % N( carbamide )145.

38 X 10-3L2, L15,SP7, Z78,SP7K, +N357.10 X 10-3565.69 X 10-5Phosphorus( TSP )142.37 X 10-2( all intervention )L2, L15,SP7, Z78,SP7K, +N35

562.13 X 10-4K( MOP )149.45 X 10-335

562.45 X 10-4


10 Non-destructive sampling

Non-destructive sampling was conducted throughout the experiment to supervise the works growing. Sampling was conducted one time every hebdomad until harvest which was on the tenth hebdomad. So a sum of 10 observations were made.


1 Plant tallness

Plant tallness was measured utilizing a measurement tape. The unit of measuring used was in centimetre.

3.10.2 Entire figure of foliages

The figure of foliages was recorded for each works harmonizing to its intervention.

3.10.3 Chlorophyll contents ( standard curve? ? )Chlorophyll content was measured utilizing SPAD metre ( Minolta SPAD Meter 502 ) . Three different degrees of foliage verdancy were measured with the SPAD metre. The values measured must be within a certain scope. Dark green is in the scope of 40-50, light viridity is between 20-40 and xanthous is below 20.

For every foliage, the chlorophyll content was measured in 3 different topographic point. The norm of the readings was taken and recorded.

3.11 Harvesting Paddy works.

Paddy workss were harvested after 70 yearss. Before the concluding crop, non-destructive sampling was conducted. Each polyethylene bag was cut at its side to reap the Paddy works. The Paddy works was so separated between the foliages and its roots.The roots with dirt were placed under running H2O to rinse away the dirt.

The length of the roots were foremost measured and so weighed to acquire the fresh weight. The whole root for each intervention was removed to undergo ARA trial. The remainder of the roots were incubated in the oven at temperature of 60EsC for 4 yearss.For the foliages, after the fresh weight was taken, they were put inside a petri dish harmonizing to their intervention.

They were so incubated in an oven with temperature of 60EsC.Dry weight of both parts ( foliages and root ) of the Paddy works was taken over several yearss. 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 changeless.

3.12 Determination of Protein Content

Leafs of each replicate for every intervention were grinded and filtered to acquire the foliage infusion.

0.1 milliliter of leaf infusion were placed into trial tubings and left for 10 proceedingss at room temperature. Then, 2 milliliter of D solution was added into the trial tubing and left for another 10 proceedingss. Finally 0.2 milliliter of folin-phenol solution was added and left for 30 proceedingss before OD reading at 600 nanometer was taken utilizing spectrophotometer.The finding of standard curve of protein uses the reagent 1mg/ml Bovine Serum Albumine: 5mg in 5ml H2O ( 1Aµ/Aµl ) .



A2 % Na2CO3 in 0.1 N NaOHBacillus1 % NaK Tartarate in dH2OC0.5 % CuSO4.5H2O in dH2OCalciferol48ml of A + 1ml of B + 1ml of CTocopherolPhenol reagent ; 1 folin-phenol:1 distilled H2O

3.13 Statistical Analysis

Appendix material frm m & A ; mTable 3.2.1: Chemicals for Okon medium




BufferK2HPO46.0KH2PO44.0AMgSO4.7H2O0.2NaCl0.1CaCl2.2H2O0.02NH4Cl1.0DL-Malic Acid5.0NaOH3.0Yeasy Extract0.1BacillusFeCl30.0110 milliliterCNaMoO4.2H2O0.00210 milliliterMnSO4.H2O0.002ZnSO4.7H2O0.00002Cu ( NO3 ) 2.3H2O0.00004H3BO30.0028pH6.8Table 3.2.2: Chemicals for RC agar



K2HPO40.5MgSO4.7H2O0.2NaCl0.1Yeast infusion0.5FeCl3.6H2O0.015DL-Malic acid5.0KOH4.8Agar15.0pH7.0Table 3.2.3: Chemicals for Nfb semisolid Malate media




DL-Malic acid5.0K2HPO40.5MgSO4.7H2O0.2NaCl0.1CaCl2.2H2O0.02*Minor component2.0*BTB2.0*Fe EDTA4.0*Vitamin1.0KOH4.0Agar1.75pH6.8*Minor component



NaMoO4.2H2O1.0MnSO4.H2O1.5H3BO31.4CuSO4.5H2O0.4ZnSO4.7H2O0.12*BTB ( Bromo Thymol Blue )


g/L ( 0.2N KOH )




Fe EDTA0.164*Vitamin





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