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IS 14807 (2000): Phosphate Solubilising Bacterial Inoculant
(PSBI) [FAD 7: Soil Quality and Gertilizers]
“!ान $ एक न’ भारत का +नम-ण”
Satyanarayan Gangaram Pitroda
““IInnvveenntt aa NNeeww IInnddiiaa UUssiinngg KKnnoowwlleeddggee””
“!ान एक ऐसा खजाना > जो कभी च0राया नहB जा सकता हहहहै””ै”
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“Knowledge is such a treasure which cannot be stolen”
IS 14807: 2000
Indian Standard
PHOSPHATE SOLUBILISING BACTERIAL
INOCULANT (PSBI) - SPECIFICATION
ICS 65.0.80
<OBIS 2000
BUREAU OF INDIAN STANDARDS
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002
October 2000 Price Group2
ANNEX C
AMENDMENT NO.1 JUNE 2008
TO
IS 14807 : 2000 PHOSPHATE SOLUBILISING
BACTERIAL INOCULANT (PSBI) - SPECIFICATION
(Page I, clau;e 4.1, line 2) - Substitute'108,for' 107'.
(Page 1,clause 4.2, line 6) - Substitute'150 to 212 Il (72 to 100 mesh)
IS sieve'for'100 J.l sieve'.
(FAD 7)
ReprographyUnit.SIS,NewDelhi,India
AMENDMENT NO. 2 MAY 2012
TO
IS 14807 : 2000 PHOSPHATE SOLUBILISING BACTERIAL
INOCULANT (PSBI) — SPECIFICATION
[Page 1, clause 6.1(h)] — Substitute ‘Net quantity in kg and area meant
for;’ for ‘Net mass in kg and area meant for;’.
(FAD 7)
Reprography Unit, BIS, New Delhi, India
SoilQualityandImprovementSectionalCommittee,FAD27
FOREWORD
ThisIndianStandardwasadoptedbytheBureauofIndianStandards,afterthedraftfinalizedbytheSoilQuality
andImprovementSectionalCommitteehadbeenapprovedby theFoodandAgricultureDivisionCouncil.
Thephosphatesolubilisingbacterialinoculants(PSBI)includedifferentgroupsofsoilmicro-organismswhich
convert insoluble phosphatic compounds into soluble form. The species of Pseudomonas, Micrococcus,
Bacillus, Flavobacterium are active in bioconversion.The mostefficientbacterial isolates are identifiedas
Pseudomonasstriata, Pseudomonasrathonis, Bacilluspolymyxa.Theseefficientmicro-organismshaveshown
capabilitytosolubilisephosphoruscontentsuppliedthroughfertilizerapplicationinthesoil.Manybrandsof
PSBIinoculantsaremarketedinthecountryandtheyhavebeenfoundtovaryinquality. Hence,theneedwas
feltforIndianStandardnotonlytotestthequalityofinoculantsinordertoprovidethefarmerswithcertified
inoculantsbutalsotoproducersimprovequalityoftheirproducts.
It is obligatory on the part of the manufacturers to employ qualified Soil MicrobiologistIMicrobiologist/
AgriculturalGraduates/Graduatesin Biologytrainedinsoil microbiology in theirstaff. Manufacturersshall
alsomaintainaqualitycontrollaboratorycapableofcarryingoutthetestsaccordingtothisstandard.
Forthepurposeofdecidingwhetheraparticularrequirementofthisstandardiscompliedwith,thefinalvalue,
observedorcalculated,expressingtheresultofatestoranalysis,shallberoundedoffinaccordancewithIS2:
1960'Rules for roundingoff numericalvalues(revised)'. The numberof significantplacesretainedin the
roundedoffvalueshouldbe thesameasthatofthespecifiedvalueinthisstandard.
IS 14807:2000
Indian Standard
PHOSPHATE SOLUBILISING BACTERIAL
INOCULANT (PSBI) - SPECIFICATION
1 SCOPE 4.4 When tested by the method prescribed in
Annex BofIS8268,thepHofPSBIshallbe6.5to7.5.
This standard prescribes the requirements, method of
sampling and tests for Phosphate Solubilising 4.5 When tested by the method prescribed in
Bacterial inoculants. Annex C, PSBI shall have phosphate solubilising
capacity intherangeofminimum 30percentinterms
2 REFERENCE
of zone formation minimum 10 mm solubilisation
The following Indian Standard contains provisions zone in a prescribed solid medium having at least 3
which, through reference in this text, constitutes mmthickness.
provisionsofthisstandard. Atthetimeofpublication,
4.6 Specified mother culture beobtained from any
the edition indicated was valid. All standards are
recognized institution maintaining themotherculture.
subjecttorevision, andpartiestoagreemen~sbasedon
The manufacturer maycontrol thequality ofbrothas
this standard are encouraged to investigate the
given inAnnex D.
possibility of applying the most recent edition of the
standard indicated below: NOTE- AtpresentNationalBiofertilizerDevelopmentCentre
(NBDC). Ghaziabad and its six Regional Centres located at
ISNo. Title Bangulore, Bhubaneshwar, Hissar. lmphal, Jabalpur and
Nagpur; Indian Agricultural Research Institute OARI), New
8268 : 1986 Specification or Rhizobium Delhi; Tamil Nadu Agricultural University (TNAU).
Coimbatore:University ofAgriculturalScience. Bangalore are
inoculant (firstrevision)
sourcesforsupplyingthemotherculture.
3 TERMINOLOGY
5 PACKING
3.1 For the purpose of this standard the following
PSBIshallbepackedinpolyethylenepacks,thickness
definitions shall apply.
of whichshall notbeless than 1()()(micron).
3.1.1 Phosphate SolubiLising Bacterial lnoculants
6 MARKING
(PSBf)
6.1 Each polyethylene pack shall be marked legibly
PSBIisaproduct havinghighpopulationofastrain(s)
andindelibly withthefollowing information:
ofbacteria intended tosolubilise insolublephosphate
a) Name of the product, specially as Phosphate
ofthesoil.
Solubilising Bacterial inoculant;
3.1.2 GroupofCrops b) Name andaddress of themanufacturer;
PSBI is beneficial for all types of crops in terms of c) Crop(s) forwhich intended;
solubilisationoffixed phosphate ofsoil.
d) Type ofthecarrierused;
4 REQUIREMENTS e) Batch number;
4.1 When tested by the method prescribed in f) Dateof manufacture;
7
Annex A of the standard, PSBI shall contain 10 g) Expiry date which shall not be less than 6
viable Phosphate Solubilising Bacterial cells/g of the months fromthedateof manufacture;
carriermaterial ondry massbasis. h) Net massin kgandarea meantfor;
4.2 PSBI shall be carrier based. colour depending on j) Storage instructions worded as under
the colour of the carrier. Carrier material such as "STORE INCOOL PLACE AWAY FROM
peat, lignite, charcoal or similar material may be DIRECT SUNLIGHTAND HEAT"; and
used. Itshall be neutralized withcalcium carbonate k) Any other information required under the
and thensterilized. When tested itshall passthrough Standards of Weights and Measures (Pack
100)lsieve. agedCommodities) Rule, 1977.
4.3 PSBI when tested by the method prescribed in 6.2 Items(c),(0and(g)shallbeprintedonacoloured
Annex A, shall have no contamination with other inkbackground
micro-organismsat lOS dilution.
IS 14807 :2000
6.3 DirectionforuseofPSBIshall beprinted briefly or producers may be obtained from the Bureau of
onthepacketsasgiveninAnnexEofthestandard. A IndianStandards.
separatepamphlet maypreferably begiven withit.
6.5 Storage
6.4 DIS CertificationMarking PSBIshallbestoredbythemanufacturerinacooland
dry place away from direct heat preferably at a
The product mayalso be marked with the Standard temperatureof20°Candnotexceeding300C. Itshall
Mark. also bethe duty of the manufacturer to instruct the
retailers and, in turn, the users about the precautions
6.4.1 TheuseoftheStandard Mark isgovernedbythe tobetakenduring storage.
provisions of Bureau ofIndian Standards Act, 1986
7 SAMPLING
and Rules and Regulations made thereunder. The
details of conditions under which the licence for the The representativesamplesofthePSBIshallbedrawn
useofStandardMarkmaybegranted tomanufacturer as prescribed inAnnexFof IS8268.
ANNEX A
(Clauses4.1 and 4.3)
DETERMINATIONOFNUMBEROFPHOSPHATESOLUBILISINGBACTERIALCELLS
A-I APPARATUS A-3 PREPARATION OF SERIAL DILUTION
FORCELLCOUNTMETHOD
A-I.I Pipettes- Graduated, 1mland 10ml.
Dispense 30 gof PSBI in270 mlof sterile waterand
A-t.2 ConicalFlasks - 150mland 250ml.
shake for 10minutes on a reciprocal shaker. Make
7
A-I.3 Screw capped tubes of 10mlcapacity. serial dilutions up to 10 level. Pipette out 0.2 ml
5 7
aliquotsof10 to10 dilutionanddeliveritonthepetri
A-l.4 Incubator
dishes containing set medium as described in
A-l.5 Petri Dishes A-2.1. Spread thealiquots over the plate. Invert the
platesandplacethem intheincubator at28±20Cfor
A-I.6 Hot AirOven 3days. Use3replicates of 105, 106 and 107dilution.
A-I.7 Autoclave
A-4 COUNTING
A-I.8 pH meter
Count the total number of colonies on the plates
A-2 REAGENTS including colonies with solubilization zone with the
helpofacolony counter.
A-2.1 Usea mediumof thefollowing composition:
Glucose 10.0g A-5 METHODS FOR COUNTING
Tri-calciumphosphate 5.0 g SOLUBILIZATIONZONES
Ammonium sulphate 0.5 g
a) Take 10gof PSBI(BF) in90 mlinwater.
Magnesium sulphate 0.1g
7
Sodiumchloride 0.2 g b) Make atenfolddilution series upto 10 .
5 7
Yeastextract 0.5 g c) Take 0.2 ml aliquots of 10 to 10 dilution
Manganese sulphate Trace using sterile pipettes and delivered to petri
FerroussuIphate Trace dishescontaining Pikowskeyi media.
Distilled water 1000 mi d) Spread it uniformly. Invert theplate~and in
Agar 15.0g cubate them upto 2weeks at28 ±2 C.
pH adjusted to7±0.2
e) Count thecolonies showing hallowcones and
A-2.3 Sterilisingandpreparation processforplatesis measure their diameter. Minimum acceptable
thesameasdescribed inIS8268. zoneis 10mmindiameter.
2
IS 14807 :2000
ANNEX B
(Clause4.4)
DETERMINATIONOFpHAND MOISTUREPERCENTAGE
8-1 pH
Make suspension of 20 g of the PSBI into 50 ml of B-2 DETERMINATION OF MOISTURE OF
distilled water and shake on a rotary shaker for 2 h. BIOFERTILIZERPACKETS(METHOD)
Filter this suspension and determine the pH of the I
Heat 109ofsamplefor 12-16hoursinanairovenat
filtratewiththehelpofpH meter.
lOO-105°C. Coolinadesiccatorandweigh. Theloss
in weight represents the moisture. Calculate the
moisture percentage on air dry weight basis, by
multiplyingthelossinweightbyten.
ANNEX C
(Clause4.5)
DETERMINATIONOFSOLUBLEPHOSPHORUS USING ASCORBIC ACID
C-O PRINCIPLE C-3.1.1 Take 20 g of ammonium molybdate and
dissolve in300mlofdistiliedwater.
Soluble phosphorus forms hetropoly
molybdophosphate complex with molybdate ions Addslowly450mlof 10NH2S04.
which on reduction produces a characteristic blue
Cooltheabovemixtureandadd 100mlof0.5percent
colour measuredat840to880nm.
solutionofantimony potassium tartrate.
Considering the higher stability of theascorbic acid, Cooland makethevolumetoone litre.Storeinglass
easiness to handle, higher tolerance to the bottleawayfromdirectsunlight.
concentrationofinterferingions,possibilitiestouseit
with all types of acids and higher stability of the C-3.2 Preparationof Mixed Reagent
developed colour (from 10to60 min),ascorbic acid
Add 1.5g of L-ascorbic acid in 100mlof theabove
insteadofstanous chloride isnow-a-days usedasthe
stock solution and mix. Add 5 mlof thissolutionto
reducing agent for the hetropoly molybdophosphate
complex formed by the soluble phosphate ions on developcolour. Mixedreagentistobepreparedfresh
asitdoes notkeepformorethan24h.
additionofammonium molybdatesolution.
C-3.3 Procedure
c-i
APPARATUS
C-3.3.1 Weigh the required material in a 100 ml
Spectrophotometer capable of transmission conicalflask.
measurements at840to880nm.
C-3.3.2 Add 50 ml of extractant and shakeitfor
Extractant: It isolsenextract. 30minonarotaryshaker.
C-2 REAGENTS C-3.3.3 FilterthesuspensionthroughWhatmanfilter
paperNo.40. Ifthefiltrateiscolouredthenaddatea
C-2.1 Ammonium Molybdate spoonofDarco-60(activatedphosphorusfreecarbon),
[(NH4)6M07024.4H20] reshakeandfiIter.
C-2.2 L-Ascorbic Acid C-3.3.4 Take a known aliquot (5 to 25 m1) of the
extract ina50mlvolumetricflask.
C-2.3 p-Nitrophenol
C-3.3.S Add 5 drops ofp-nitrophenol indicator (1.5
percent solution in water) and adjust the pH of the
C-2.4 4NHzS04
extract between 2 and 3 with the help of 4NH2S04.
C-3 PREPARATIONOFREAGENTS The yellowcolour willdisappear whenthepH ofthe
solutionbecomes 3. Swirlgentlytoavoid lossofthe
C-3.1 SulphomolybdlcAcid solutionalongwiththeevolution ofC04.
3
