Schedule II

PART-B (See Section 22) 1. A. METHOD OF ANALYSIS OF RHIZOBIUM BIO-FERTILISERS 1. Apparatus 1.1. Pipettes Graduated 1 ml and 10 ml 1.2. Dilution Bottles or Flasks 1.3. Petri Dishes Clear, Uniform, flat-bottomed. 1.4. Hot - Air Oven Capable of giving uniform and adequate temperature equipped with a thermometer, calibrated to read up to 250 C and with vents suitably located to assure prompt and uniform heating. 1.5. Autoclave 1.6. Incubator 1.7. Hand Tally or Mechanical counting Device 1.8. PH meter 2. Reagents 2.1. Congo Red and one percent aqueous solution 2.2. Medium Use a plating medium of the following composition Agar20 g Yeast Extract1 g Mannitol10 g Potassium hydrogen phosphate (K2HP04)0.5 g Magnesium sulphate (MgS04 7H20)0.2 g Sodium Chloride (NaCL)0.1 g Congo red2.5 ml Distilled water1000 ml PH7.0 2.3. Sterilising and preparation procedure for plates: 2.3.l. Sterilise the sampling and plating equipment with dry heat in a hot air oven at not less than 160°C for not less than 2 hours. 2.3.2. Sterilise the media by autoclaving at 120°C for 20 min. To permit passage of steam into and from closed containers when autoclaved, keep stoppers slightly loosened. Air from within the chamber of the steriliser should be ejected allowing steam pressure to rise. PREPARATION OF PLATING MEDIUM AND POURING 2.3.3. Prepare growth medium in accordance with the composition indicated in 2.2. 2.3.4. Melt the required amount of medium in boiling water or by exposure to following steam in partially closed container but avoid prolonged exposure to unnecessarily high temperature during and after melting. Melt enough medium which will be used with in 3h. Re-sterilisation of the medium may cause partial precipitation of ingredients. 2.3.5. When holding time is less than 30 min, promptly cool the melted medium to about 45°C, and store until used, in a water bath or incubator at 43 to 45°C. Introduce 12 to 15 ml of liquefied medium or appropriate quantity depending on size of the petridish at 42 to 44°C into each plate. Gently lift the cover of the dish just enough to pour in the medium, Sterilise the lips of the medium containers by exposure to flame. a. immediately before pouring. b. periodically during pouring, and c. when pouring is complete for each batch of plates, if portions of melted medium remain in containers and are to be used without subsequent sterilization for pouring additional plates. As each plate is poured thoroughly mix the medium with test portions in the Petri dish. 2.3.6. By rotating and tilting the dish and without splashing the medium over edge, spread the medium evenly over the bottom of the plate. Provided conditions so that the medium solidifies with reasonable promptness (5-10 min) before removing the plates from level surface. 3. PREPARATION OF SERIAL DILUTIONS FOR PLATE COUNTS 3.1 Dispense 30 g of Inoculant to 270 ml of sterile distilled water and shake for 10 min on a reciprocal shaker. Make serials dilutions up to 10 Take 0.2 ml or suitable aliquots of 10 to 10 dilutions using sterile pipettes and deliver to Petri dishes containing set medium as given in 2.1 and spread it uniformly. Invert the plates and promptly place them in the incubator. 4. INCUBATION OF PLATES 4.1. Label the plates and incubate at 28 +/-2°C for 3 to 5 days for fast growing Rhizobia and 5 to 10 days slow growing ones 4.2. Colony Counting aids. Count the colonies with the aid of magnifying lens under uniform and properly controlled artificial illumination. Use a colony counter, equipped with a guide plate and rules in centimetre square. Record the total number of colonies with the hand tally. Avoid mistaking particles of un-dissolved medium or precipitated matter in plates for pin-point colonies. The distinguish colonies form dirt, specks and other foreign matter, examine doubtful objects carefully. 4.3. Count all plates but consider for the purpose of calculation plates showing more than 30 and less than 300 colonies per plate. Disregard colonies which absorb Congo red and stand out as reddish colonies. Rhizobium stands out as white, translucent, glistening and elevated colonies. Count such colony numbers and calculate figures in terms of per litre, of carrier. Also check for freedom from contamination at 10 dilution. 1. B. METHOD OF ANALYSIS OF AZOTOBACTOR BIO-FERTILISER 1. Apparatus- same as 1 of 1A of part B of schedule-II. 2. Reagents: 2.1. Medium Use a plating medium of the following composition Agar20 g Sucrose (C12 H2 Ol1)20.0 g Ferric sulphate (Fe2 (S04)2)0.1 g Dibasic potassium phosphate (K2HP04) 1.0 g Magnesium sulphate (MgS04, 7H20) 0.5 g Sodium Chloride (NaCl)0.5 g Calcium carbonate (CaC02)2.0 g Sodium Molybdate (Na2 MO 04)0.005 gm Distilled water PH 1000 ml 6.8 to 7.2 2.2. Sterilising & preparation procedure for plates: Same as 2.3.1 and 2.3.2 of 1 A of Part B of schedule-II PREPARATION OF PLATING MEDIUM AND POURING Same as 2.3.3, 2.3.4, 2.3.5 & 2.3.6 of 1 A of part B schedule-II. 3. PREPARATION OF SERIAL DILUTIONS FOR PLATE COUNTS 3.1 Dispense 30 g of Inoculant to 270 ml of sterile distilled water and shake for 10 min on a reciprocal shaker. Make serials dilutions up to 10 Take 0.2 ml or suitable aliquots of 10 to 10 dilutions using sterile pipettes and deliver to Petri dishes containing set medium as given in 2.1 and spread it uniformly. Invert the plates and promptly place them in the incubator. 4. INCUBATION OF PLATES:- 4.1. Label the plates and incubate at 28+/ - 3°C for 4 to 6 days. 4.2. Colony counting aids: Same as 4.2 of 1 A of Part B of Schedule-II Azotobactor chrococcum colonies are gummy raised with or without striations, viscous and often sticky. The pigmentation varies from very light brown to black. Count the colony number and observe the cyst formation as given below and calculate number per gram of the carrier material. Grow the vegetative cells at 30 C on Burks agar medium comprising sucrose 20 g, di-potassium hydrogen phosphate 0.64 g, dihydrogen potassium phosphate 0.20 g; sodium chloride 0.20 g; calcium sulphate 0.05 g, sodium molybdate 0.001 g; ferric sulphate 0.003 g. agar 20 g and distilled water 1.000 ml. Look for vegetative cells after 18 to 24 h either by simple staining method or through a phase contrast microscope. Grow the cyst cells on Burks agar medium as given above with 0.3 percent n-butanol in place of the carbon source. Look for cyst formation after 4 to 5 days incubation. 1.C. METHOD OF ANALYSIS OF AZOSPIRILLUM BIO-FERTILISER 1. Apparatus - same as 1 A of Part B of schedule II 2. Reagents 2.1. Medium Use a plating medium of the following composition Malic acid5.0 g Potassium hydroxide4.0 g Di-potassium hydrogen phosphate 0.5 g Ferrous sulphate0.05 g Manganese sulphate0.01 g Magnesium sulphate0.1 g Sodium chloride0.2 g Calcium chloride0.1 g Sodium Molybdate0.002 g Distilled water1000ml Boromothymol blue (0.5% alcoholic solution) 2.0 m. Agar1.7 g pH adjusted to6.5 - 7.0 2.2.Sterilising & preparation procedure for plates : Same as 2.3.1 and 2.3.2 of 1 A of Part B of schedule - II PREPARATION OF PLATING MEDIUM AND POURING Same as 2.3.3, 2.3.4, 2.3.5 & 2.3.6 of 1 A of part B of schedule - II. 3. PREPARATION OF SERIAL DILUTIONS FOR PLATE COUNTS: Same as 1 B of part B of schedule-II 4. Incubation of Plates :- 4.1. Label the plates and incubate at 28+/ - 3°C for 4 to 6 days. 4.2. Colony counting aids: Same as 4.2 of 1 A of part B of schedule-II Counting Counting the tubes or plates which have turned blue in colour after inoculation and ascertain the presence of pellicles in undisturbed medium. To determine usual contamination on the same examine doubtful objects carefully. Count all plates/ tubes which have turned blue and consider them for the purpose of calculation. Count such type of tubes/ plates and tally this count with MPN table Annexure-E to get the number of cells per gram of the carrier. Azospirillum Count/g of carrier = MPN table value x Dilution level Dry mass of product 1. D. MEDTHOD OF ANALYSIS OF PHOSPHATE SOLUBLISING BACTERIAL BIO-FERTILISER 1. Apparatus. - same as 1 A of Part B of schedule-I 2. Reagents. - 2.1. Medium Use a plating medium of the following composition: Glucose10.0 g Tri-calcium phosphate5.0 g Ammonium sulphate0.5 g Magnesium sulphate0.1 g Sodium Chloride0.2 g Yeast extract0.5 g Manganese sulphateTrace Ferrous sulphateTrace Distilled water1000 ml Agar15.0 g PH adjusted to 7 +/- 0.2 2.2. Sterilising & preparation procedure for plates: Same as 2.3.1 and 2.3.2 of 1 A of Part B of schedule-II preparation of plating medium and pouring: Same as 2.3.3, 2.3.4, 2.3.5 & 2.3.6 of 1 A of part B of schedule-II. 3. PREPARATION OF SERIAL DILUTION FOR PLATE COUNTS: Same as 1 B of part B of schedule-II 4. Incubation of Plates:- 4.1. Label the plates and incubate at 28+/ - 3°C for 4 to 6 days. 4.2. Colony counting aids: Same as 4.2 of 1 A of part B of schedule - II Counting Count the total number of colonies on the plates including colonies with solubilisation zone with the help of a colony counter. Methods for counting solubilisation zones (a) Take 10 g of PSBI (BF) in 90 ml in water (b) Make a ten fold dilution series up to 10 (c) Take 0.2 ml aliquote of 10 to 10 dilution using sterile pipettes and delivered to Petri dishes containing pikowskeyi media. (d) Spread it uniformly, Invert the plates and incubate them up to 2 weeks at 28+/- 2°C. (e) Count the colonies showing hallow cones and measure their diameter. Minimum acceptable zone is 10 mm in diameter. GUIDELINES OF MAINTENANCE AND PREPARATION OF CULTURE AND QUALITY CONTROL AT BROTH STAGE 1. Rhizobium: 1. Maintenance of pure cultures 1.1. Maintain pure cultures of rhizobia on yeast extract mannitol agar (YEMA) slants of the following composition. Mannitol10.0 g Potassium hydrogen phosphate (k2HP04) 0.5 g Magnesium sulphate (MgS04 7H20) 0.2 g Sodium chloride (NaCl)0.1 g Calcium Carbonate (Ca C03)1.0 g Yeast extract1.0 g Agar18.0 g Distilled water1 liter pH6-8-7.0 1.2. Transfer a loopful of the pure culture to each of the slants aseptically in an inoculation room and incubate at 28+/- 2°C for 3 to 10 days depending upon the species of Rhizobium. Always keep pure cultures at 4°C. 2. PREPARATION OF INOCULUM CULTURES 2.1. Prepare yeast monnitol broth of the composition as given in 1.1. minus the agar. 2.2. Transfer a loopfull of the culture into a 100/250 ml conical flasks containing the broth. Incumbate the flasks at 28+- 2°C on the rotary shaker for 2 to 6 days. 4. QUALITY CONTROL TESTS RECOMMENDED AT BROTH STAGE : 4.1. Qualitative Tests 4.1.1. Check for freedom from visible contaminants 4.1.2. The pH of the bacterial broth shall normally be between 6.5 and 7.5 4.1.3. Smear and Gram stain 4.1.3.1. Reagents a. Ammonium oxalate crystal violet stain weigh 0.2 g of crystal violet and dissolve in 20 ml of 95 percent ethyl alcohol. Dissolve separately 0.8 g of ammonium oxalate in 80 ml of distilled water. Mix the two solutions and fitter through a filter paper. b. Iodine solution Iodine1.00 g Potassium Iodide 2.00 g Distilled water 300 ml Weight the ingredients and dissolve in water. Filter through a filter paper. c. Erythrosine Erythrosine 1.00 g Phenol5.00 g Distilled water 100 ml Weight the ingredients, dissolve in distilled water and filter through a filter paper. 4.1.3.2. Procedure Prepare a smear on a claim microscope slide, fix over a flame by gentle and intermittent heating, air cool and flood with ammonium oxalate crystal violet stain for 1 min. After removing the excess of ammonium oxalate crystal violet, wash the slide under a gentle stream of running tap water. Flood the slide with iodine solution for half of minute remove excess stain wash with 95 percent ethyl alcohol and finally wash under a gentle stream of running tap water. Flood the slide with erythrosine stain for about 3 min, wash under a gentle stream of running tap water and dry between the folds of a filter paper. Examine the slide under a compound microscope using an oil immersion objective. Note:- A smear prepared from undiluted broth should be free from Gram positive cells. The presence of a few gram positive cells in occasional fields which may be due to dead cells in the medium maybe disregarded. 4.1.4. Absence of Growth on Glucose -Peptone Agar The composition of the glucose - peptone agar is as follows: Glucose10.0 g Peptone20.0 g Sodium chloride (NaCI)5.0 Agar (IS 6850}15.0 Distilled water1000 ml Bromocresol purple10 ml of ethyl alcohol solution1.6 persons pH7.2 Note: When a loopful of the broth is streaked into this medium and incubated at 28+/- 2°C for 24 h, the purple violet colour of the medium (due to the indicator bromocresol purple) shall not change. If the colour changes to yellow (acidic reaction) or blue (alkaline reaction) the broth is grossly contaminated Hence, the broth should be rejected. 4.1.5. Streak on yeast Extract monnitol Agar with Congo Red When a loopful of broth culture is streaked to plate of this medium and incubated at 28 +/- 2°C for 3 to 10 days, it shall show colonies of bacteria with growth characteristics same as that of the pure culture use in the preparation of the broth, Other wise, the broth should be rejected. 4.2. Quantitative Test 4.2.1. Viable or Plate Counts Serially dilute one millilitre of the broth to obtain dilutions of the order of to 10 . Plate 0.2 ml aliquots of the dilutions on YEMA plates and incubate at 28 +/- 2°C for 2 to 6 days, depending on the species of Rhizobium. The counts of viable Rhizobium in the final broth from shake culture or fermentors shall be not less than 10 to 10 cells/ml. Other wise, the broth should be rejected. 2. AZOTOBACTOR 1. Maintenance of pure cultures 1.1. Maintain pure cultures of Azotobactor on slants of the following composition Agar20 gm Sucrose20 gm Ferric Sulphate0.1 Dibasic Potassium Phosphate 1.0 gm Magnesium Sulphate0.5 gm Calcium carbonate2.0 gm Sodium Molybdate0.005 gm Distilliled water1000 ml PH6.8 to 7.2 1.2. Transfer a loopful the pure culture to each of agar slants aseptically in an inoculation room and incubate at 28 +/- 2°C for 3 to 10 days depending upon the species of Azotobactor. Always keep culture pure cultures at 5°C. 2. Preparation of inoculum culture 2.1 Prepare Jonsons media broth of the composition as given in 1.1. minus the agar 2.2. Transfer a loop full of the culture into 100ml/250 ml, conical flask containing the broth. Incubate the flasks at 28 +/- 2°C on a rotary shaker for 2 to 6 days. 3. Quality control Tests recommended at broth stage 3.1. Qualitative test. 3.1.1 Check for free from contaminants by preparing slide and observing under microscope. 3.1.2 The pH by bacterial broth shall normally be between 6.5 to 7.0 3.1.3 Gram staining test shall be carried out as described in 4.1.3, 4.1.3.1 and 4.1.3.2 of Rhizobium of this standard. 3.2. Quantitative test 3.2.1. Viable cell count: same as 4.2. of IB of part B of Schedule II 3. AZOSPIRILLUM 1. Maintenance of pure cultures 1.1. Maintenance of pure cultures of Azospirillum on nitrogen free bromothymol blue medium and maintain as semi solid medium as described in 2.1 of this standard 1C of part B of Schedule II 1.2. Transfer a loopful of pure culture to each of the agar culture tube aseptically in an inoculation room and incubate 28 +/- 2°C for three days and keep in undisturbed. Always keep pure culture below 5°C. 2. Preparation of Inoculum culture: Inoculum culture shall be prepared as described in 2.1, 2.2 of Rhizobium of this standard. 3. Quality Control Test recommended at Broth Stage 3.1. Qualitative Test 3.1.1. Check for free from contaminants by preparing slide and observing under microscope. 3.1.2. The pH of bacterial broth shall normally be between 7.0 to 8.0. 3.1.3. Gram staining test shall be carried out as described in 4.1. 3., and 4.1.3.1 & 4.1.3.2. of Rhizobium of this standard. 3.1.4. See the colour change in the media after 24 hours from inoculation. The colour will change from green to blue. 3.1.5. Watch the pellicle just below the surface of the media. It checked on the third day after keeping inoculated broth undisturbed. 3.2. Quantitative Test 3.2.1. Most Probable Number (MPN) as given in Annexure-E. The counts of Azosprillium in the final broth from shake culture or fermentors shall be not less than 10 to 10 cells / ml. Otherwise the broth should be rejected. 4. PHOSPHATE SOLUBILIZING BACTERIAL INOCULANT (PSBI) 1. Maintenance of pure cultures 1.1. Maintain pure culture of PSBI on the medium as described in 2.1 of 1 D of part B of schedule- II in the form of slants 1.2. Transfer a loopful of pure culture to each of the agar slants aseptically in an inoculation room and incubate at 28 + /- 2°C for three days. Always keep pure culture below 5°C. 2. Preparation of Inoculum culture: Inoculum culture shall be prepared as described in 2.1, 2.2. of Rhizobium changing the media composition as mentioned in 2.1 of this standard. 3. Quality control test recommended at Broth Stage 3.1. Qualitative Test 3.1.1. Check for free from visible contaminants by microscope and observing solubilisation zones. 3.1.2. The pH of bacterial broth shall normally be between 6.5 to 7.0. 3.1.3. Gram staining test shall be carried out as described in 4.1.3,4.1.3.1, and 4.1.3.2 of Rhizobium. 3.2. Quantitative Test 3.2.1 Viable cell count serially dilute one millilitre of broth plate 0.2. ml aliquots of the dilutions on pikowkyasi media (as given in 2.1 of 1 D of Part B of Schedule II) plates and incubate at 28 +/- 2°C for 2 to 6 days. The counts of PSBI in the final broth from shake culture or fermentors shall be not less than 108 to 109 cells/ml. Otherwise, the broth should be rejected ANNEXURE-A DETERMINATION OF pH 1. Make suspension of 20 g of the ASI into 50 ml of distilled water and shake on a rotary shaker for 2 hours. Filter this suspension and determine the pH of the filterate with the help of pH meter. ANNEXURE-B DETERMINATION OF MOISTURE OF BIO-FERTILISER PACKETS (METHOD) 2.1. Heat 10 g of sample for 12-16 hours in an air over at 100-105°C, in desiccator and weigh. The loss in weight represents the moisture. Calculate the moisture percentage on air dry weight basis, by multiplying the loss in weight by ten. ANNEXURE-C DETERMINATION OF SOLUBLE PHOSPHORUS USING ASCORBIC ACID. Principle - Soluble phosphorus from hetropoly molybdophosphate complex with molybdate ions which on reduction produces blue colour measured at 840 to 880 nm. Considering the higher stability of the ascorbic acid, easiness to handle, higher tolerance to the concentration of interfering ions, possibilities to use it with all type of acids and higher stability of the developed colour (10 to 60 min), ascorbic acid instead of stannous chloride is now-a-days used as the reducing agent for the hetropoly molybdophosphate complex formed by the soluble phosphate ions on addition of ammonium molybdate solution. apparatus - Spectrophotometer capable of transmission measurements at 840 to 880 nm. Reagents - Ammonium Molybdate (NH4) 6 M07024 4H20) I - Ascorbic acid P - Nitrophenol 4NH2S04 Preparation of Reagents - Sulphomolybdic acid. Take 20 g of Ammonium molybdate and dissolve in 300 ml of distilled water Add slowly 450 ml of 10 N2 S04. Cool the above mixture and add 100 ml of 0.5 percent solution of Antimonypotassium tartrate. Cool and make the volume to one litre. Store in glass bottle away from direct sunlight. Preparation of mixed Reagent - Add 1.5 gm of L - ascorbic acid in 100 ml of the above stock solution and mix. Add 5 ml of this solution to develop colour. Mixed regent is to be prepared fresh as it does not keep for more than 24 h. Procedure - Weigh the required material in a 100 ml conical flask. Add 50 ml of extractant and shake it for 30 min on rotary shaker. Filter the suspension through whatman filter paper No. 40. If the filterate is coloured then add a tea spoon of Darco-60 (activated phosphorus free carbon) reshake and filter. Take a known aliquot (5 to 25 ml) of the extract in a 50 ml volumetric flask. Add 5 drops of P-nitrophenol indicator (1.5 percent solution in Water) and adjust the pH of the extract between 2 and 3 with the help of NH2S04. The yellow colour will disappear when the pH of the solution becomes 3. Swirl gently to avoid loss of the solution along with the evolution of C04. When the C02 evolution has subsided. Wash down the neck of the flask and dilute the solution to about 40 ml. Add 5 ml of the sulphomolybdic acid mixed reagent containing ascorbic acid Swril the content and make up the volume. Measure the transmission after 30 min. at 880 nm using red filter. The blue colour developed remains stable up to 60 minutes. Record the concentration of Phosphorus (P) in the extract from the standard curve and calculate the concentration of soluble phosphorus as follows. Calculation - (a) Weight of the substance taken = xg (b) Volume of the extractant added=50 ml (c) Volume of the extract taken for p=y ml determination. (d) Volume made after colour developed = 50 ml (e) Reading from the standard curve = z ppm against percent transmission recorded. (f) Soluble phosphorus. Percent p = ZX50X10 -6X5O ________________X 100 Y,X Preparation of standard curve - Prepared standard curve using 0.1 to 0.6 ppm P in 50 ml volumetric flask plot the standard curve by taking concentration of soluble P on x-axis and percent T on Y-axis using a semi-log graph paper. It is a straight line relationship between the soluble P and percent T when plotted on semi-log graph paper. ANNEXURE-D DIRECTION FOR USE OF BIO FERTILISERS Rhizobium 1. The contents of the packet are sufficient for seeds to be sown in the area indicated on the package. 2. Use only for the leguminous crops mentioned, before the expiry date and do not expose to direct sun light or heat. 3. Mix the inoculants with the seeds gently with the minimum amount of water, taking care to avoid damage to seed coat. Dry the inoculated seeds under shade over clean paper or gunny bag and sow immediately. This is not a chemical fertiliser and hence do not mix inoculated seeds or RI with chemical fertilisers. Azotobactor 1. Use only for the non-leguminous crops and before the expiry date. 2. The contents of the packet are sufficient for seeds to be sown in 0.4 hectare. 3. This is not a chemical fertilizer, hence do not mix it or the inoculated planting material with chemical fertilizers or pesticides. 4. Use for the crops specified on the packet. 5. Do not expose to direct sunlight or heat. Azospirillum 1. The contents of the packet are sufficient enough to the given area to be broadcasted or given seedlings for root dipping depending on the specified crops as denoted in the packet. Mix the inoculants with seeds gently with the minimum amount of water, taking care to avoid damage to seed coat. Dry the inoculated seed under shade over clean surface gunny bag and sow them immediately. 2. Use only for the crops mentioned paddy, Azospirillum brasillense for other crops like millets, etc use crop specific strain. Use before the expiry date and do expose to direct sun light or heat. 3. ASI is not a chemical fertilizer hence do not mix inoculated seeds or ASI with agrochemicals. PHOSPHATE SOLUBILISING BACTERIA 1. The contents of the packet are sufficient enough to the given area to be broadcast or given seedlings for root dipping depending on the specified crops as denoted on the packet. Mix the inoculants with seeds gently with the minimum amount of water, taking care to avoid damage to seed cost. Dry the inoculated seed under shade over clean dry paper organic bag and sow them immediately. 2. In order to solubilise fixed soil phosphate use PSBI for all type of crops. Use before the expiry date and do not expose to direct sunlight or heat. 3. PSBI is not chemical fertiliser hence do not mix inoculated seeds or PSBI with agrochemicals. T. MADAN MOHAN REDDY, Secretary to Government, Legislative Affairs & Justice, Law Department.