The Telangana Bio-fertilisers (Monitoring and Quality Control) Act, 2006

1. NITROGEN FIXING BIO-FERTILISERS:a. RHIZOBIUM INOCULANTS (RI) (IS 8268 - 2001)

i. RI shall be carrier based, the colour depending on

the colour of the carrier.

ii. RI shall contain a minimum of 107 viable Rhizobium

cells/g of the carrier on dry - mass basis till 6 months expiry period from the date of manufacture. The number shall be counted by the plate count method as given in 4.2 and 4.3 of 1A of Part B of Schedule - II.

iii. RI shall have a maximum of six months expiry

period from the date of manufacture.

iv. RI shall have no contamination with other micro-

organisms at 105 dilution when counted as given in 4.3 of 1A of Part B of Schedule – II.

v. The pH of RI shall be between 6.5 and 7.5 when

tested as given in Annexure- A.

vi. RI shall show effectiveness nodulation on all those

species and / or cultivars on the packet before the expiry date when tested. If good effectiveness pink nodulation is obtainable in the inoculated species together with total absence or sometimes presence of stay nodules in the controls, it should be concluded that RI contains effective Rhizobium. The total dry mass of inoculated plants shall be significantly higher than that of the un-inoculated controls and at least 50 percent more than the controls.

vii. The carrier material such as peat, lignite, peatsoil,

humus favouring growth to be neutralized with calcium carbonate and sterilized shall be in the form of a powder capable of passing through 150 to 212 micron (72 to 100 mesh) IS sieve.

viii. Specified mother culture be obtained from any

recognized institution maintaining the mother cultures. The manufacturer may control the quality of the broth, it should get verified at least by two institutions as mentioned below:

Note: At present National Bio-fertiliser Development centre (NBDC), Ghaziabad and its six Regional Centres located at Bangalore, Bhubaneshwar, Imphal, Hissar, Jabalphur and Nagpur, Indian Agricultural Research Institute (IARI), New Delhi, Tamil Nadu Agricultural University (TNAU) Coimbatore, University of Agricultural Science, Bangalore are sources for supplying the mother culture.

ix. The RI carrier shall be in the form of fribal (moist) with 30-40 percent (m/m) moisture content when tested as given in Annex-B.

1. (b) AZOTOBACTER CHROOCOCCUM INOCULANTS (AI) (IS 9138 - 2002) i. AI shall be carrier-based, the colour depending on the colour of the carrier. ii. AI shall contain a minimum of 107 viable Azotobacter cells/g of the carrier on dry - mass basis till 6 months expiry period from the date of manufacture. The number shall be counted by the plate count method as given in 4.2 of 1 A of part B of Schedule II and Azotobactor Chroococcum clolonies 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 Part B - Schedule II (4.2) and calculate number per gram of the carrier material. iii. AI shall have a maximum of six months expiry period from the date of manufacture. iv. AI shall have no contamination with other microorganisms at 105 dilution. v. The pH of AI shall be between 6.5 and 7.5 when tested as given in-A. vi. The carrier material such as peat, lignite, peatsoil, humus favouring growth to be neutralized with calcium carbonate and sterilized shall be in the form of a powder capable of passing through 150 to 212 micron (72 to 100 mesh) IS sieve. vii. Specified mother culture be obtained from any recognized institution maintaining the mother cultures. The manufacturer may control the quality of the broth, it should get verified atleast by two institutions as mentioned in VIII of 1 of part A of Schedule-I. viii. The AI carrier shall be in the form of fribal (moist) with 30-40 percent (m/m) moisture content when tested as given in Annexure-B. C. AZOSPIRILLUM INOCULANTS (ASI) (IS 148062000) i. ASl shall contain 107 viable Azospirillum cells / g of the carrier material on dry mass basis. ii. ASl shall have no contamination with other microorganisms at 105 dilution. ASl contamination in semi solid medium should be checked by semi dilution and spread plate method with solid complete medium. iii. The pH of ASI shall be 6.5 to 7.5 when tested as given in Annexure-A. iv. The ASl carrier shall be in the form of fribal (moist) with 30-40 percent moisture. When tested as given in Annexure-B. v. ASl shall show effective root development on all cultivars / crops against which the inoculant is intented to be used. vi. Specified mother culture be obtained from any institution maintaining the mother cultures. The manufacturer may control the quality of the broth, it should get verified atleast by two institutions as mentioned in VIII of I of Part A of Schedule - I. 2. PHOSPHATE SOLUBILISING BACTERIAL INOCULANT (PSBI) (IS 14807-2000) i. PSBI shall contain 107 available phosphate solubilising bacterial cells / g of the carrier material on dry mass basis. ii PSBI shall be carrier based colour depending on the colour of the carrier. Carrier material such as peat, lignite charcoal may be used. It shall be neutralized with calcium carbonate and then sterlised. When tested it shall pass through 100 micron sieve. iii. PSBI shall have no contamination with other microorganisms at 105 dilution. iv. The pH of PSBI shall be 6.5 to 7.5. v. The PSBI carrier shall be in the form of fribal (moist) with 30-40 percent (m/m) moisture content when tested as given in Annexure-B. vi. PSBI shall have phosphate solubilising capacity in the range of minimum 30 percent in terms of zone formation minimum 10 mm solubilisation zone in a prescribed solid medium having at least 3 mm thickness, when tested by the method prescribed in Annexure-C. vii Specified mother culture be obtained from any recognized institution maintaining the mother culture. The manufacturer may control the quality of broth, it should get verified at least by two institutions mentioned in VIII of I of Part A of Schedule-I.

1. RHIZOBIUM:

1. Packing:

RI shall be packed material of low density polyethylene / polypropylene bags thickness of which shall be 75-100 micron minimum.

2. Marking:

Each packet shall be marked legibly to give the following information:

i. Name of the product, specifically as Rhizobium

inoculants.

ii. Leguminous crop for which intended.

iii. Name and address of the manufacturer. iv. Type of the carrier. v. Batch or code number. vi. Date of manufacture. vii. Date of expiry (agreed between the manufacturer

and the purchaser subject to minimum 6 months from the date of manufacture).

viii. Net quantity and the area meant for. ix. Storage instructions worded as under : STORE IN

COOL PLACE AWAY FROM DIRECT SUN AND HEAT.

x. Any other information.

3. Item (ii), (vi) and (vii) shall be printed on a colured

ink background.

4. Directions for the use of RI shall be printed briefly on

the packet as given in Annexure D. A Separate pamphlet may preferably be given with it.

5. The product may also be marked with the standard

mark.

6. The use of the standard mark is governed by the

provisions of the Bureau of Indian Standards Act, 1986 and the rules and regulations made thereunder. The details of conditions under which the licence for the use of the standard Mark may be granted to manufacturers or producers may be obtained from the Bureau of Indian Standards.

7. RI shall be stored by the manufacturer in a cool and dry place away from direct heat preferably at a temperature of 150 C to 300 C. It shall also be the duty of the manufacturer to instruct the retailers and, in turn the users about the precautions to be taken during storage.

2. AZOTOBACTOR: Packing:

1. AI shall be packed in packaging material of low

density polythene / polypropylene bags thickness of which shall be 75-100 micron minimum.

Marking:

2. Each packet shall be marked legibly to give the

following information,-

a. Name of the product, specifically as Azotobactor

inoculant;

b. Non-leguminous crop for which intended; c. Name and address of the manufacturer; d. Type of the carrier; e. Batch of code number; f. Date of expiry (agreed between the manufacturer

and the purchaser subject to minimum 6 months from the date of manufacture);

g. Net quantity and rate of application;

h. Storage instructions worded as under STORE IN

COOL PLACE AWAY FROM DIRECT SUN AND HEAT;

i. Any other information.

3. Directions for the use of AI shall be printed briefly on

the packet as given in Annexure – D. A separate pamphlet may preferably be given with it.

4. The product may also be marked with the standard

mark.

5. The use of the standard mark is governed by the

provisions of the Bureau of Indian Standards Act, 1986 and the rules and regulations made thereunder. The details of conditions under which the licence for the use of standard mark may be granted to manufacturers or producers may be obtained from the Bureau of Indian Standards.

6. AI shall be stored by the manufacturer in a cool and

dry place away from direct heat preferably at a temperature of 150C to 300C. It shall also be the duty of the manufacturer to instruct the retailers and, in turn, the users about the precautions to be taken during storage.

Act No.63 of 1986.

3. AZOSPlRILLIUM Packing:

1. ASI shall be packed in polyethylene packs, thickness

which shall not be less than 75 - 100 micron.

Marking:

2. Each polyethylene packs shall be marked legibly

and indelibly with the following information,-

a. Name of the product, specially as Azospirlllum

inoculant.

b. Name and address of the manufacturer. c. Crop (S) for which intended. d. Type of the carrier used. e. Batch number. f. Date of manufacture. g. Expiry date which shall not be less than 6 months

from the date of manufacture.

h. Net mass in kg and area meant for. i. Storage instructions worded as under: STORE IN

COOL PLACE AWAY FROM DIRECT SUNLIGHT AND HEAT.

j. Any other information required under the Standards

of Weights and Measures (packaged commodities) Rules 1977.

3. Direction for use of ASI shall be printed briefly on the

packets as given in Annexure-D of the standard. A separate pamphlet may preferably be given with it.

4. The product may also be marked with the Standard

Mark.

5. The use of the Standard Mark is governed by the

provisions of Bureau of Indian Standards Act, 1986 and rules and regulations made thereunder. The details of conditions under which the licence for the use of standard

mark may be granted to manufacturer or producers may be obtained from the Bureau of Indian Standards.

6. ASI shall be stored by the manufacturer in a cool and dry place away from direct heat preferably at a temperature of 200C and not exceeding 300C. It shall also be the duty of the manufacturer to instruct the retailers and, in turn, the users about the precautions to be taken during storage.

4. PHOSPHATES SOLUBULISING BACTERIAL INOCULANT (PSBI)

Packing:

1. PSBI shall be packed in polyethylene packs,

thickness of which shall not be less than 100 micron.

Marking:

2. Each polyethylene packs shall be marked legibly

and indelibly with the following information,-

a. Name of the product, specially as Phosphate

solubilising Bacterial inoculants.

b. Name and address of the manufacturer. c. Crop (S) for which intended. d. Type of the carrier used. e. Batch number. f. Date of manufacture.

g. Expiry date which shall not be less than 6 months

from the date of manufacture.

h. Net mass in kg and area meant for. i. Storage instructions worded as under: STORE IN

COOL PLACE AWAY FROM DIRECT SUNLIGHT AND HEAT.

j. Any other information required under the Standards

of Weights and Measures (packaged commodities) Rules 1977.

3. Direction for use of PSBI shall be printed briefly on

the packets as given in Annexure - D of the standard. A separate pamphlet may preferably be given with it.

4. The product may also be marked with the Standard

Mark.

5. The use of the Standard Mark is governed by the

provisions of Bureau of Indian Standards Act, 1986 and rules and regulations made thereunder. The details of conditions under which the licence for the use of standard mark may be granted to manufacturer or producers may be obtained from the Bureau of Indian Standards.

6. PSBI shall be stored by the manufacturer in a cool

and dry place away from direct heat preferably at a temperature of 200C and not exceeding 300C. It shall also be the duty of the manufacturer to instruct the retailers and, in turn the users about the precautions to be taken during storage.

1. General Requirements:

1.0. In drawing, preparing and handling the samples,

following precautions and directions shall be observed,-

1.1. Sampling shall be carried out by a trained and

experienced person as it is essential that the sample should be representative of the lot to be examined.

1.2. Since the samples are also required for micro-

biological analysis, utmost care is necessary to avoid extraneous contamination while drawing and handling the samples and to preserve them in their original conditions till they are ready for examination in the laboratory.

1.2.1. No preservations or bactericidal / fungicidal

agent shall be added to samples required for microbiological analysis.

1.3. Samples in their original un-opened packets

should be drawn and sent to the laboratory. This will prevent possible contamination of the samples during handling and also help in revealing the true condition of the material.

1.4. Intact packets shall be drawn from a protected

place not exposed to dampness, air, light, dust or soot and transferred to clean containers.

1.5. The sampling appliances and sample containers

shall be sterile.

1.6. All precautions shall be taken to protect the sample of the material being sampled, the sampling instruments and the sample container against adventitious contamination at the time of drawing the sample, opening containers and transferring the samples. 2. Sampling Equipment:

2.1. A suitable scoop made of stainless steel may be

used for drawing samples.

2.2. The sampling equipment shall be perfectly clean

and sterile. It shall be properly sterlised by heating in a hot air oven at 1600 C for not less than 2h or by autoclaving for not less than 20 min at 1200 C and held in suitable containers to prevent re-contamination.

3. Scale of Sampling: 3.1. Lot

All units (containers in a single consignment of type of material belonging to the same batch of manufacture) shall constitute a lot. If a consignment consists of different batches of the manufacture the containers of the same batch shall be separated and shall constitute a separate lot.

3.2. Batch An inoculant prepared from a batch fermentor or a

group of flasks (containers) constitute a batch.

3.3. For ascertaining conformity of the material to the requirements of the specification, samples shall be tested from each lot separately.

3.4. The number of packets to be selected from a lot

shall depend on the size of the lot and these packets shall be selected at random and in order to ensure the randomness of selection.

4. Drawal of samples:

4.1. Three (3) samples should be drawn separately

from each lot as per 1.3 of part A of Schedule - II. One sample should be sent for analysis, one has to be handed over to the dealer under acknowledgement, and one will be treated as referee sample.

4.2. The samples should be put in a cloth bag which

may be sealed as specified in Form K along with other details like sample no. / code no. which enables its identification.

1. A. METHOD OF ANALYSIS OF RHIZOBIUM BIOFERTILISERS

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

2500C and with vents suitably located to assure prompt and uniform heating.

1.5. AutocIave. 1.6. Incubator. 1.7. Hand Tally or Mechanical counting Device. 1.8. pH meter.

2. REAGENTS

2.1. Congo Red - one percent aqueous solution. 2.2. Medium. Use a plating medium of the following composition

Agar 20 g Yeast Extract 1 g Mannitol 10 g Potassium hydrogen phosphate (K2HP04) 0.5 g Magnesium sulphate (MgS04 7H20) 0.2 g Sodium Chloride (NaCL) 0.1 g Congo red 2.5 ml Distilled water 1000 ml pH 7.0

2.3. Sterlising and preparation procedure for plates:

2.3.1. Sterlise the sampling and plating equipment with

dry heat in a hot air oven at not less than 1600 C for not less than 2 hours.

2.3.2. Sterlise the media by autoclaving at 1200 C for 20

min. To permit passage of steam into and from closed containers when autoclaved, keep stoppers slightly loosened. Air from with in the chamber of the sterliser 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 flowing steam in partially closed container but avoid prolonged exposure to unnecessarily high temperature during and after melting. Melt enough medium which will be used within 3h.

Re-sterlisation 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 450 C, and store until used, in a water bath or incubator at 43 to 450 C. Introduce 12 to 15 ml of liquified medium or appropriate quantity depending on size of the petridish at 42 to 440 C into each plate. Gently lift the cover of the dish just enough to pour in the medium, Sterlise 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. Provide 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 or demineralized water and shake for 10 min on a reciprocal shaker or homogeniser. Make serial dilutions up to 1010. Take 0.2 ml or suitable aliquots of 106 to 109 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 +/- 20 C for 3 to 5 days for fast growing Rhizobia and 5 to 10 days for 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 centimeter 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. To 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 108 dilution.

1. B. METHOD OF ANALYSIS OF AZOTOBACTOR BIOFERTILISER

1. APPARATUS: same as 1 of 1 A of part B of Schedule - II. 2. REAGENTS: 2.1. Medium

Use a plating medium of the following composition

Agar 20 g Sucrose (C12 H22 O11) 20.0 g Ferric sulphate (Fe2 (S04)2) 0.1 g Dibasic potassium phosphate (K2HP04) 1.0 g Magnesium sulphate (MgSO4, 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 1000 ml pH 6.8 to 7.2

2.2. Sterlising & 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:

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 1010 Take 0.2 ml or suitable aliquots of 106 to 109 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 +/- 30 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 300 C on Burks agar medium comprising sucrose 20 g, dipotassium 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 AZOSPIRlLLUM BIOFERTILISER

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 acid 5.0 g Potassium hydroxide 4.0 g Di-potassium hydrogen phosphate 0.5 g Ferrous sulphate 0.05 g Manganese sulphate 0.01 g Magnesium sulphate 0.1 g

Sodium chloride 0.2 g Calcium chloride 0.1 g Sodium Molybdate 0.002 g Distilled water 1000 ml Boromothymol blue 2.0 ml (0.5 % alcoholic solution ) Agar 1.7 g pH adjusted to 6.5 - 7.0

2.2. Sterlising & 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 +/- 30 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. METHOD OF ANALYSIS OF PHOSPHATE SOLUBULISING 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:

Glucose 10.0 g Tri-calcium phosphate 5.0 g Ammonium sulphate 0.5 g Magnesium sulphate 0.1 g Sodium Chloride 0.2 g

Yeast extract 0.5 g Manganese sulphate Trace Ferrous sulphate Trace Distilled water 1000 ml Agar 15.0 g pH adjusted to 7 +/- 0.2

2.2. Sterlising & 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 I B of Part B of Schedule – II

4. INCUBATION OF PLATES: 4.1. Label the plates and incubate at 28 +/- 30 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 107 c. Take 0.2 ml aliquote of 105 to 107 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 +/- 20 C.

e. Count the colonies showing hallow cones and

measure their diameter. Minimum acceptable zone is 10 mm in diameter.

GUIDELINES ON 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.

Mannitol 10.0 g Potassium hydrogen phosphate (k2HP04) 0.5 g Magnesium sulphate (MgSO4 7H20) 0.2 g Sodium chloride (NaCl) 0.1 g Calcium Carbonate (Ca CO3) 1.0 g Yeast extract 1.0 g Agar 18.0 g

Distilled water 1 litre pH 6.8-7.0

1.2. Transfer a loopful of the pure culture to each of the

agar slants aseptically in an inoculation room and incubate at 28 +/- 20 C for 3 to 10 days depending upon the species of Rhizobium. Always keep pure cultures at 40C.

2. Preparation of Inoculum Cultures:

2.1. Prepare yeast mannitol 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. Incubate the flasks at 28

20C on a rotary shaker for 2 to 6 days.

3. Quality Control Tests Recommended at Broth Stage: 3.1. Qualitative Tests

3.1.1. Check for freedom from visible contaminants 3.1.2. The pH of the bacterial broth shall normally be

between 6.5 and 7.5

3.1.3. Smear and Gram stain 3.1.3.1. 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 filter through a filter paper.

b. Iodine solution

Iodine 1.00 g Potassium Iodide 2.00 g Distilled water 300 ml

Weigh the ingredients and dissolve in water. Filter through a filter paper.

c. Erythrosine

Erythrosine 1.00 g Phenol 5.00 g Distilled water 100 ml

Weigh the ingredients, dissolve in distilled water and filter through a filter paper.

3.1.3.2. Procedure

Prepare a smear on a clean 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 a 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 may be disregarded.

3.1.4. Absence of Growth on Glucose - Peptone Agar The composition of the glucose - peptone agar is as

follows:

Glucose 10.0 g Peptone 20.0 g Sodium chloride (NaCl) 5.0 Agar (IS 6850) 15.0 Distilled water 1000 ml Bromocresol purple ethyl alcohol solution

10 ml of 1.6 persons

pH 7.2

Note:- When a loopful of the broth is streaked into this medium and incubated at 28 +/- 20 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.

3.1.5. Streak on yeast Extract mannitol Agar with Congo Red

When a loopful of broth culture is streaked to a plate of this medium and incubated at 28 +/- 20 C for 3 to 10 days, it shall show colonies of bacteria with growth characteristics same as that of the pure culture used in the preparation of the broth. Otherwise, the broth should be rejected.

3.2. Quantitative Test 3.2.1. Viable or Plate Counts

Serially dilute one milliliter of the broth to obtain dilutions of the order of 106 to 109. Plate 0.2 ml aIiquots of the dilutions on YEMA plates and incubate at 28 +/- 20 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 108 to 109 cells / ml. Otherwise, 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

Agar 20 gm Sucrose 20 gm Ferric Sulphate 0.1 Dibasic Potassium Phosphate 1.0 gm Magnesium Sulphate 0.5 gm Calcium carboantare 2.0 gm Sodium Molybdate 0.005 gm Distilliled water 1000 ml pH 6.8 to 7.2

1.2. Transfer a loopful of the pure culture to each of

agar slants aseptically in an inoculation room and incubate at 28 +/- 20 C for 3 to 10 days depending upon the species of Azotobactor. Always keep culture pure cultures at 50 C.

2. Preparation of inoculum culture

2.1. Prepare Jensons media broth of the composition

as given in 1.1 minus the agar

2.2. Transfer a loop full of the culture into a 100ml/ 250

ml, conical flask containing the broth. Incubate the flasks at 28 + /- 20C 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 3.1.3, 3.1.3.1 and 3.1.3.2 of Rhizobium of this standard.

3.2. Quantitative test 3.2.1. Viable cell count: same as 3.2. of 1B of Part B of

Schedule II

3. AZOSPIRILLUM

1. Maintenance of pure cultures:

1.1. Maintain pure culture 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 +/- 20 C for three days and keep it undisturbed.

Always keep pure culture below 50 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 3.1.3., and 3.1.3.1 & 3.1.3.2. 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 is 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 108 to 109 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 +/- 20 C for three days. Always keep pure culture below 50 C. 2. Preparation of Incoculum 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 3.1.3, 3.1.3.1 and 3.1.3.2 of Rhizobium.

3.2. Quantitative Test:

3.2.1. Viable cell count serially dilute one milliliter 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 +/- 20 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 - 1050 C, in a 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 forms 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 types of acids and higher stability of the developed colour (10 to 60 min), ascorbic acid instead of stanous 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 MO7O24 4H2O) I - Ascorbic acid P - Nitrophenol 4NH2SO4

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 N2SO4. cool the above mixture and add 100 ml of 0.5 percent

solution of Antimony potassium 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 reagent 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 corbon) 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 4NH2SO4. 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 CO4.

When the CO2 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 Swirl 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 extractrant add = 50 ml c) Volume of the extract taken for p determination.

= y ml

d) Volume made after colour developed = 50 ml e) Reading from the standard curve against percent transmission recorded.

= z ppm

f) Soluble phosphorus.

Percent p =

Zx50x10-6x50

x 100 Y, X

Preparation of stardard 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 semilog graph paper.

ANNEXURE - D DIRECTIONS 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.

4. 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 cops like millets, etc use crop specific strain. Use before the expiry date and do not expose to direct sun light or heat.

3. ASI is not a chemical fertilizer hence do not mix

inoculated seeds or ASI with agro-chemicals.

PHOSPHATE SOLUBILISING BACTERIA.

1. The contents of the packet pre 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 coat. 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 agro-chemicals.