Processing of Garbage Decomposting at Ditch House, Saswad.

• Every day the garbage collected from 800kg to 1 tonne of garbage comes in the ditch house for the fertilizer process.

 

 

• After the final segrigation is done. and wet waste & dry waste garbage are classified.

• Again spraying the compost culture on the wet waste garbage and keep it for 1 day for airration.because it does not smell the garbage.

• After one day the entire wet waste garbage is out into the ecoman machine. for the garbage fertilizer process and early decomposting process.

• Ecoman machine should continue for 5 to 10 min after 2 hours a day.

• Again give the 5 to 6 machine a rotation per day. becacuse all the garbage in the machine drum rotates all the waste and mixes together.
• After the machine was run for 2 days, the complete waste material colour becomes dark chocklate & blackish .& fine compost fertilizer are prepared.

• The machine was run from 30 to 40 oC temperature to go to waste thermophilic stage in the machine. Because all the pathogen, insect ,lazy , smell & alage to die.

• After then take out all the garbage compost prepared in the machine & cut out the machine & make it’s bed’s or piles.

• After reading the temperature of the bed’s or piles. until the bed’s or piles do not come to the cooling stage.
• Every 400 to 500 kg compost is prepared from 1 tonne of wet waste garbage in 2 day’s.
• From waste disposal in the machine, it takes only 20 to 25 days from prepared compost bed’s. & the good fertilizer is ready.

isolation of phosphate solubilizing bacteria :

Phospahte is a limiting nutrient in most of the soils because it does not remain in soluble form. It reacts with calcium abundantly available in soli to form calcium phosphate and becomes insoluble. Action of certain bacteria can dissolve rock phosphate and make the phosphate available slowly in soil. This action  mainly through formation of organic acids from fermentable sugars and cellulose.

Pikovskaya (PVK) medium :

Ingredient	Conc. g/L
Glucose	10
Ca3(PO4)2	5
(NH4)2SO4	0.5
NaCl	0.2
MgSO4  H2O	0.1
KCl	0.2
Yeast Extract	0.5
MnSO4	0.002
FeSO4 7H2O	0.002
pH	7.5
Agar for solidification	15

• Isolation of phosphate solubilising bacteria :

1. Dilute serially the liquid culture enrichment to 10^-4 in sterile NaCl Buffer.
2. Streak all four dilutions individually on four different plates of Pikovskaya (PVK)    and incubate the plates at 37 °C for 3-5 days.
3. Observe the plates and colonies that form zone of clearance surrounding the colonies caused by dissolution of Calcium Phosphate in the medium. Although most of the colonies formed are of those able to dissolve calcium phosphate a s source, those with larger zone are because of an acid formation that enhances phosphate Solubilization
4. Select well isolated colony and that dissolves Calcium phosphate. Suspend in sterile physiological saline. Dilute serially and plate out again for further purification. At times cultures that do not solubilize rock phosphate also grow near an active culture using the phosphate dissolved by the neighbor.
5. Incubate plates for 3-5 day till similar colonies with zone of clearance is seen.
6. Perform microscopy from a suspension of colony for purity test
7. Transfer the colony on the same medium for maintenance
8. Cultures can be maintained by pouring sterile liquid paraffin of the well grown agar slant.

phosphate solubilizing bacteria

 

n

 

 

production of trichoderma

Trichoderma viride  : is a fungus and a biofungicide.

It is used for seed and soil treatment for suppression of various diseases caused by fungal pathogens.

Fungi are the most important biological agents against plant pathogens. Trichoderma species are common filamentous fungi, capable of hyper parasitizing pathogenic fungi and are highly efficient antagonists.   It is also highly competitive, displaying antagonism against other pathogenic fungi. It has been successfully cultivated for use as a biofungicide, with Trichoderma harzianum, T. viride, and T. hamatum as the most common species used for biological control.

Benefits :

• Is environmentally friendly.
• Leaves no chemical residue.
• Reduces crop losses and increases yields.
• Is usable on many crops and usable against many pathogenic fungi.
• Is inexpensive to purchase and is easy and inexpensive to produce.
• Is a potential source of income.

 

Mass production of Trichoderm:

Materials required :

• • sorghum/ jawar.
  • Mother Culture (may be procured from NICL
    biocontrol laboratory)
  • 8″ x 12 ” plastic bag
  • Cotton
  • Rubber band

• • Plastic pipe of 1 1⁄2 inch length and 1 1⁄2 cm – 2 cm diameter
    having both side open.
  • Pressure cooker of 5 Lts. or above
  • Heating system (gas/electric heater)
  • Stone/wood
  • Fresh Water/ distilled water.
  • spoon.

procedure :

• Take 1 kg of sorghum/Jawar in the poly pack and add 2 % sucrose solution & soak it. (if grains contain dust then wash it twice before adding fresh water).
• Place the plastic pipe in the middle of the plastic pack (opening end) in such a way that level of the pipe and plastic remain equal.
• Tie it with the help of rubber band. Plug the opening end of the pipe tightly with the help of the cotton.
• Cover the cotton plug with a paper using rubber band.
• Place the thick paper inside the pressure cooker surrounding the cooker wall.
• Place the stone in the cooker and add water into the cooker just below the stone.
• Place the plastic pack inside the cooker and put it on heating system. Wait until 3 times gas release from the cooker (3 whistles). Remove the packet from the cooker until totally cool down.

Inoculation method :

• Place a burner at the laminar air flow  and wait for 3-4 min.
• Wash hand and the spoon with ethenol.
• Open the paper cover from the plastic pack.
• Take mother culture (Talc based) by using opposite end of the spoon and pour it in to the plastic pack, removing cotton plug in front of burner.
• Plug it again and keep the plastic pack in incubator @ 37 c for 10-12 days. The entire grain based medium will turn green due to sporulation of Trichoderm

Precautions :

• Do not open the cotton plug until use.
• Keep it in a cold place (refrigerator preferably after sporulation)
• Avoid direct sunlight until use.

onion plantation & RBD ( randomised blog design ) of bio-treatment inoculation with bactericide & fungicide.

onion plantation :

onion plantation is done in the 1.25 R area field.with replication of bio treatment inoculation of bactericide & fungicide.

Bio treatment inoculation of fungicide & bactericide :

1) Inoculation of mycorrhizal fungi treatment :

• Arbuscular mycorrhizas (AM) are generally beneficial associations (symbioses) between plant roots and specialised soil fungi.
• High AM colonisation indicates good soil and crop health.
• AM associations can be the dominant pathway for uptake of phosphorus (P) and zinc (Zn), but are also important for other nutrients.
• Nearly all crop plants can form AM associations except lupins and brassicas such as canola.
• Crop sequences  which have extended periods without AM plants can reduce nutrient uptake and yield of following host crops.
• Arbuscular mycorrhizas (AM) are generally beneficial associations (symbioses) between plant roots and specialized soil fungi.
• all the AM fungi in any particular soil will be able to colonize all the plants that are grown there.
• symbiosis is based on exchange of nutrients: the fungus increases the ability of host roots to take up nutrients while the plant provides the fungus with sugars.

2) Inoculation of  bacillus treatment ( bactericide ) :

• Bacillus is a genus of gram-positive, rod-shaped bacteria and a member of the phylum Firmicutes.
• Bacillus species can be obligate aerobes  or facultative anaerobes (having the ability to be aerobic or anaerobic).
• They will test positive for the enzyme catalase when there has been oxygen used or present.
•  Bacillus includes both free-living (nonparasitic) and parasitic pathogenic species. Under stressful environmental conditions, the bacteria can produce oval endospores that are not true ‘spores’, but to which the bacteria can reduce themselves and remain in a dormant state for very long periods.
• Many species of Bacillus can produce copious amounts of enzymes which are made use of in different industries.
• Some species can form intracellular inclusions of polyhydroxyalkanoates under certain adverse environmental conditions, as in a lack of elements such as phosphorus, nitrogen, or oxygen combined with an excessive supply of carbon sources.

bacillus culture broth 

Agarose gel Electrophoresis :

What is gel electrophoresis?

Electrophoresis is a technique commonly used in the lab to separate charged molecules, like DNA, according to size.

• Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA^?, RNA^? and proteins^?according to their size.
• Charged molecules move through a gel when an electric current is passed across it.
• An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge.
• The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end (opposites attract!).
• The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it.
• Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result the molecules are separated by size.

How is gel electrophoresis carried out?

Preparing the gel

• Agarose gels^? are typically used to visualise fragments of DNA. The concentration of agarose used to make the gel depends on the size of the DNA fragments you are working with.
• The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA are separated on higher concentrations of agarose whilst larger molecules require a lower concentration of agarose.
• To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until all of the agarose powder has melted.
• The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to make wells for the sample to be pipetted into.
• The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. The buffer conducts the electric current. The type of buffer used depends on the approximate size of the DNA fragments in the sample.

Preparing the DNA for electrophoresis

• A dye is added to the sample of DNA prior to electrophoresis to increase the viscosity of the sample which will prevent it from floating out of the wells and so that the migration of the sample through the gel can be seen.
• A DNA marker (also known as a size standard or a DNA ladder) is loaded into the first well of the gel. The fragments in the marker are of a known length so can be used to help approximate the size of the fragments in the samples.
• The prepared DNA samples are then pipetted into the remaining wells of the gel.
• When this is done the lid is placed on the electrophoresis tank making sure that the orientation of the gel and positive and negative electrodes is correct (we want the DNA to migrate across the gel to the positive end).

Separating the fragments

• The electrical current is then turned on so that the negatively charged DNA moves through the gel towards the positive side of the gel.
• Shorter lengths of DNA move faster than longer lengths so move further in the time the current is run.
• The distance the DNA has migrated in the gel can be judged visually by monitoring the migration of the loading buffer dye.
• The electrical current is left on long enough to ensure that the DNA fragments move far enough across the gel to separate them, but not so long that they run off the end of the gel.

 

Visualising the results

• Once the DNA has migrated far enough across the gel, the electrical current is switched off and the gel is removed from the electrophoresis tank.
• To visualise the DNA, the gel is stained with a fluorescent dye that binds to the DNA, and is placed on an ultraviolet transilluminator which will show up the stained DNA as bright bands.
• Alternatively the dye can be mixed with the gel before it is poured.
• If the gel has run correctly the banding pattern of the DNA marker/size standard will be visible.
• It is then possible to judge the size of the DNA in your sample by imagining a horizontal line running across from the bands of the DNA marker. You can then estimate the size of the DNA in the sample by matching them against the closest band in the marker.

After completion of the PCR perform the gel elctrophoresis.

• Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules likeDNA, RNA^? and proteins^?according to their size.
• Charged molecules move through a gel when an electric current is passed across it.
• An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge.
• The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end (opposites attract!).
• Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result the molecules are separated by size.

Preparing of 1 x TAE-To prepare 500 mkl of 1x TAE , add 10 ml of 50 x TAE buffer to 490 ml of sterile distiiled water.

·         Preparing the gel-(To prepare 50 ml of 0.8% agarose gel ,add 0.4 g agarose to 50 ml of 1x TAE  buffer in a glass beaker . heat the mixture on microwave until agarose dissolves completely .Add 0.5 ul ethidium bromide in it. )

• Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose used to make the gel depends on the size of the DNA fragments you are working with.
• The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA are separated on higher concentrations of agarose whilst larger molecules require a lower concentration of agarose.
• To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until all of the agarose powder has melted.
• The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to make wells for the sample to be pipetted into.
• Once the gel has cooled and solidified (it will now be opaque rather than clear) the comb is removed.
• The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. The buffer conducts the electric current. The type of buffer used depends on the approximate size of the DNA fragments in the sample.

   

  RESULT :                                                                                             

  

  

PCR /Polymerase Chain Reaction :

what is PCR ?

PCR is an abbreviation for “polymerase chain reaction”. PCR is a method for amplifying DNA. So if you have one molecule of DNA you can increase it to thousands of molecules within a few hours. This is achieved using an enzyme called DNA polymerase and the raw material or “building blocks” of DNA. The enzyme copies the original DNA molecule to make two molecules. Then it copies each of these molecules to provide a total of four molecules. Then these four molecules are copied to give a total of eight DNA molecules. And so on …

PCR is a process used to makes copies of a piece of DNA. PCR consists of three steps: Denaturation, annealing, and extension. Each PCR stage doubles the number of DNA molecules. The process is continued for many cycles to generate a huge number of copies. The process is performed on a PCR cycler or PCR machine. The PCR cycler heats or cools the PCR mixture at the appropriate time in order to allow denaturation, annealing, or extension.

• PCR is used for amplify a precise fragment of DNA from a complex mixture of starting material called template DNA
• It is very simple technique for charcaterizing ,analyzing &synthesizing DNA from any living orgnisms.
• PCR is a in vitro method of enzymtic synthesis of specific DNA fragment which developed by Kary Mullis in  1983.
• In this method there are 5 steps involved-

1. Final elongation-This step perfomed at temp. 70-74c for 5-15 min.
2. Elongation step-the temp. depends on the DNA polymerase used.Taq polymerase has its optimum activity at 75-80c .Commonly a temperature of 68-72 c is used with this enzyme.The DNA polymearse synthesizes a new DNA stand complementary to the DNA template strand by incorporating dNTPs that are complementary to the template in 5′ to 3′ direction condensing the 5′ -phosphate group of dNTPs with 3′-hydroxyl group at the end of the extending DNA starnd.
3. Annealing Step-reaction temperature is lowered to 50-65c for 20-40 sec.allowing annealing of primers to the single stranded DNA template.
4. Denaturation step-heating reaction mixture to 94-98c for 20 sec.DNA denatures due to disruption of hydrogen bonds between complementry basesof DNA starnd .
5. Initializing steps-heating the reaction mixture at 94-96 c for 1-9 min.to break the hydrogen bonds in DNA strands.

• Required components-
• DNA template that contain region to be amplified
• two primers complemntary to the 3′ ends of each of the sense and antisense strnd of the DNA
• Thermostable DNA polymeraselike taq
• dNTPs
• buffer solution provide stabilty of DNA polymerase

Procedure-(all procedure follow under freezing condition)

• Take a 12 hrs old culture of bacteria (azotobacter ,pseudomonas, Rhizobium)
• take 1000 ul mili Q water & add a single colony in it with the help of tooth pic.(dilution )
• ( we can use this dilution for next day but it kept under -80c)
• take 1 ml diluted sample in another vial
• add 5ul master mix then
• add 17 ul miliQ water then
• add 1 ul primer (forwaed)+1 ul primer ( reverse )

Operating system of PCR instrument-

 

Isolation of Bacillus from soil-

                    Bacillus is an aerobic bacteria found in soil ,water, air,food.It is a fast growing spore forming organism with a wide nutritonal capability.The ability of baccillus to form endospore help these organisns to survive adverse envionmental conditions for very long time.the spore are resistant to dessication ,heat (upm to 80c,30min)irradiation.they can be inactivated by heigher temprature(121c)chemical & solvents.

Bacillus species are involved in degradation of sevral organic compounds like starch ,celluose,priteins&lipids.Some of them produce acids that help in dissolution of calcium phosphate &mica  &play important role in plant nutrition.

sample collection :  collect rhizosphere soil sample.

Medium for isolation of soil :

Ingredient	Conc.g/l
Peptone	5.0
Sodium chloride	5.0
Beef extract	1.5
Yeast extract	1.5
PH	7.4
Agar	20

isolation of bacillus from heat treated soil :

Protocol for isolation of bacillus from soil :

 

Growth of bacillus in liquid medium :

               use 5ml nutrient medium with out agar in 25ml tubes and inoculate the growth from a fresh slant. incubate tubes at 100 rpm for 24 h till good growth occurs. transfer 5ml broth to 50 ml medium in 250ml flask and incubate at 100 rpm for 2-3 days till good growth occurs. once the vegetative growth is complete, the nutrient limitation in the medium causes the bacillus to form endospores . microscopically the spores would look as follows. Residual cytoplasm in a cell will be stained by crystal violet or safranine. spore remains as an un stained part. the spores can be terminal, sub terminal or central. they may or may not cause swelling of sporangium { the vegetative cell}   depending on the species of bacillus.

RESULT :

 

 

 

isolation of azatobactor sp.

Azotobacter  : is a genus of usually motile, oval or spherical bacteria that form thick-walled cysts and may produce large quantities of capsular slime. They are aerobic, free-living soil microbes which play an important role in the nitrogen cycle in nature, binding atmospheric nitrogen, which is inaccessible to plants, and releasing it in the form of ammonium ions into the soil (nitrogen fixation). it is used by humans for the production of biofertilizers, food additives, and some biopolymers.  Azotobacter species are Gram-negative bacteria found in neutral and alkaline soils.

jensen’s medium is recommonded for detection & culivation of nitrogen fixing bacteria.

medium composition :

Ingredient	Conc.g/l
Sucrose	20
Dipotassium phosphate	1.0
Magnesium sulphate	0.5
Sodium chloride	0.5
Ferrous sulphate	0.1
Sodium molybdate	0.005
Calcium carbonate	2.0
Ph	7.5
Agar for solidification	15

protocol for isolation of azotobacter-

 

growth of the culture in liquid medium for use as biofertilizer :

• prepare 4- 5 jensen”s liquid medium flask each containing 100ml of the medium in 500ml flask [ or 50ml in 250ml flask ] also prepare 10ml tubes in 50ml culture tubes.
• Transfer fresh growth from a slant to 10  ml medium and incubate the tubes stationary for 24 hours.
• transfer the tubes to shaker at 100 rpm for 24h or till good growth occurs.
• perform microscopy to make sure that the culture is pure .
• transfer the 10 ml liquid broth to 100 ml  medium in 250ml flask. use 5ml for 50ml medium in 250ml flask.
• transfer the inoculated flasks to shaker at 100 rpm till good growth occurs. thus may take 4-5 days.
• this broth should have above 1* 107 cells per ml as counted by colony forming units on jensens agar plates by spread plate technique.

Enrichment of soil sample in media-

RESULT :

 

 

soil sterilization :

                  Soil sterilization is the act of killing or destroying the action of diseases causing organism that are present in the soil without causing harm to other plants. soil sterilization is a process by which chemicals are used to soil living organism that are capable of destroying plants and causing disease in the soil to be inactive,These chemicals are selective in nature, that is, when they are used, they only affect particular diseases for which it is meant for, without causing harm to the other organism that inhabit the soil.

Formaldehyde / formalin  method  : 38 % – 40 % conc.

This is effective on some fungal diseases, but relatively ineffective against many pests. It is used at a strength of 1 part formalin 38 — 40% to 49 parts water (approximately 0.5 litre in 25 litres or 1 pint to 6 gallons) and the soil is thoroughly drenched — approximately 23 litres (5 gallons) being required per m² (square yard) to 23cm (9in) depth. There is a wait period of 20-40 days according to temperature (which must be sufficiently high otherwise the formaldehyde will become polymerized) before the soil can be used.

Method of Using : For soil sterilization commercial formaldehyde is diluted with water. Success depends on penetration of the dilute solution to every particle of soil at the requisite depth, its retention in the soil for an adequate length of time and thorough clearance of the fumes before potting or planting begin. Because soil does not absorb formaldehyde appreciable. it will penetrate only as far as the water that carries it. Thus, a large amount of water must be used to carry the formaldehyde well down into the subsoil. Roughly speaking, a heavy soil requires twice as much water as a light one, to wet it to a given depth.Penetration is ensured by seeing that the soil is loose, friable
and moist (potting condition). Formaldehyde should not beused when the soil temperature is 40° F. or less.Retention is almost inevitable since the fumes are given off
slowly (especially in winter) and consequently act over a long time.
In some cases, covering the soil may be beneficial.

1.  for soil sterilization commercial formaldehyde is diluted with 500 liter water to added 10 liter formaldehyde is spray in the total 1.25 R area of  field or onion plot.
2.  also field is covering with the help of polythene paper.

Randomised Block Design of onion crop plot with fungicide & bactericide tratment :

prepared fully  planning for onion plantation of  randomised block design with fungicide & bactericide bio-treatment  replication is done.

• prepared flat bed in the field for bio treatment replication of onion plantation.