The Andhra Pradesh Bio-fertilisers (Monitoring and Quality Control) Act, 2006.
**THE ANDHRA PRADESH BIO-FERTILISERS (MONITORING AND QUALITY**
**CONTROL) ACT, 2006**

**(Act No. 6 of 2006)**

**ARRANGEMENT OF SECTIONS**

**SECTIONS**

**CHAPTER - I**
**PRELIMINARY**

1. Short title, extent and commencement
2. Definitions

**CHAPTER – II**
**PRICE CONTROL**

3. Fixation of prices of Bio-fertilisers
4. Display of Stock Position and Price List
5. Issue of cash or credit memorandum
6. Control on distribution of Bio-fertilisers by manufacturers

**CHAPTER - III**
**REGISTRATION OF MANUFACTURERS / DEALERS**

7. Dealers to be registered
8. Application for Registration
9. Grant or refusal of Certificate of Registration
10. Period of validity of Certificate of Registration
11. Renewal of Certificate of Registration

**CHAPTER - IV**
**RESTRICTION ON MANUFACTURE, SALE ETC OF BIO-FERTILISERS**

12. Restriction on manufacture, sale and distribution of Biofertilisers.
13. Manufacturers to comply with certain requirements for Laboratory facility.
14. Bulk sale of Bio-fertilisers.
15. Disposal of non-standard Bio-fertilisers.
16. Manufacturers to appoint officers responsible for compliance of the Act.
17. Restriction of sale/use of Bio-fertilisers.

**CHAPTER – V**
**ENFORCEMENT AUTHORITIES**

18. Appointment of Controller and Registering Authority.
19. Appointment of Inspectors.
20. Qualification for appointment of Inspectors.
21. Powers of Inspectors.
**CHAPTER - VI**
**ANALYSIS OF SAMPLES**

22. Analysis of samples.


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23. Qualification for appointment of Bio-fertiliser Analyst in the Bio-fertiliser
Control Laboratories.
24. Time limit for analysis and communication of results.
**CHAPTER - VII**
**MISCELLANEOUS**

25. Suspension / cancellation of Registration Certificate.
26. Appeal.
27. Grant of duplicate copies of Certificate of Registration etc.
28. Amendment of Certificate of Registration.
29. Maintenance of Records and submission of returns etc.
30. Fees.
31. Offences and penalties.
32. Protection of action taken in good faith.
33. Power to remove difficulties.
34. Power to make rules.
35. Repeal of Ordinance.

**Schedule - I.**

**Schedule - II.**


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**THE ANDHRA PRADESH BIO-FERTILISERS (MONITORING AND QUALITY**
**CONTROL) ACT, 2006**

**(Act No. 6 of 2006)**

[03[rd] January, 2006]

**AN ACT TO PROVIDE FOR THE MAINTENANCE OF SPECIFIED COUNTS OF**
**VIABLE CELLS OF MICROORGANISM, VOLUME OF BIOFERTILIZER**
**AND TO REFRAIN FROM THE PRODUCTION OF CONTRABAND OR**
**SPURIOUS OR PHYTO-TOXI MATERIAL AND DESCRIPTION OF**
**SPECIES OF MICRO ORGANISM IN THE MANUFACTURE OF A**
**PARTICULAR TERMED BIO-FIRTILIZER AND TO MONITOR THE**
**EFFECTIVENESS OF THE PRODUCT IN THE LABEL BY THE**
**MANUFACTURER** **AND** **FOR** **THE** **MATTERS** **CONNECTED**
**THEREWITH OR INCIDENTAL THERETO.**

Be it enacted by the Legislature Assembly of the State of the Andhra
Pradesh in the Fifty-sixth year of the Republic of India as follows:

**CHAPTER-I**
**Preliminary**

**1. Short title, extent and commencement - (1) This Act may be called the**
Andhra Pradesh Bio-fertilisers (Monitoring and Quality Control) Act, 2006.

(2) It extends to the whole of the State of Andhra Pradesh.

(3) It shall be deemed to have come into force with effect on and from the
21st November, 2005.

**2. Definitions - In this Act, unless the context otherwise requires,-**

(1) 'Bio-fertiliser' means a inert material containing microbial inoculants
(living organism) as specified in Schedule I;

(2) 'Certificate of Source' means a certificate issued by the Government or
as the case may be, the Manufacturer to the Dealer indicating the source from
which the Bio-fertiliser for purpose of sale is obtained;

(3) 'Controller' means the person appointed as Controller of Bio-fertiliser
by the Government and includes any other person empowered by the
Government to exercise or perform all or any of the functions as may be
prescribed;

(4) 'Dealer' means a person carrying on the business of selling Biofertilisers and includes a manufacturer and an agent carrying on such
business;

(5) 'Government' means the State Government;

(6) 'Grade' means the living organism contents in Bio-fertiliser expressed
in number of cells/gram of carrier material/millilitre of liquid Bio-fertiliser;

(7) 'Inspector' means an Inspector appointed under Section 19 of the Act;


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(8) 'Label' means any written, printed or graphic matter on the package
and any other covering in which the package is placed or packed and includes
any written, printed or graphic matter accompanying the Bio-fertiliser;

(9) 'Manufacturer' means a person who produces Bio-fertilisers and
expression manufacturer with its grammatical variations shall be construed
accordingly;.

(10) 'Non-standard Bio-fertiliser' means,
(i) if its label contains any statement, design or graphic
statement/ representation thereto which is false or
misleading in any material particular, or if its package is
otherwise deceptive in respect of its contents; or

(ii) if it is an imitation of, or is sold under the name of
another Bio-fertiliser; or

(iii) if any word, statement or other information required by
or under this Act to appear on the label is not displayed
thereon in such conspicuous manner as the other words,
statements or graphic matter have been displayed on the
label and in such terms as to render it likely to be read and
understood by any ordinary individual under customary
conditions of purchase and use; or

(iv) if it is not packed and labelled as required by or under
this Act; or

(v) if it is not registered in the manner required by or under
this Act; or

(vi) if the label contains any reference to registration other
than the registration number; or

(vii) if the bio-fertiliser is not in conformity with the
prescribed standards as specified in section 2 (16) of this Act
and packed with any substance which is not included in the
registration;

(11) 'Notification' means a notification published in the Andhra Pradesh
Gazette and the word 'notified' shall be construed accordingly;

(12) 'Offer for sale' includes a reference to intimation by a person and a
person who proposed for sale, exhibit the price list, indicating the prices of Biofertiliser;

(13) 'Package' means a box, bottle, casket, tin, barrel, case receptacle,
sack, bag wrapper or other thing in which a bio-fertiliser is placed or packed;

(14) 'Premises' means any land, shop, and stall or place where any biofertiliser is manufactured or stored or sold or used and includes any vehicle
carrying bio-fertilisers;

(15) 'Prescribed' means prescribed by rules made under this Act;

(16) 'Prescribed standard' means prescribed under this Act;

(17) 'Registering Authority' means a registering authority appointed
under section 18;


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(18) 'Schedule' means the Schedule appended to this Act;

(19) 'State' means the State of Andhra Pradesh.

**CHAPTER-II**
**Price Control**

**3. Fixation of prices of Bio-Fertilisers - (1) The Government may by**
notification regulate equitable distribution and also the rates of Bio-fertilisers
in the manner as may be prescribed.

(2) The Government may having regard to the local conditions of any
area, the period of storage of Bio-fertilisers, and other relevant circumstances,
fix different prices or rates for Bio-fertilisers having different periods of storage
or for different classes of consumers.

(3) No dealer or manufacturer shall sell or offer for sale any bio-fertiliser
at a price exceeding the maximum price or rate fixed under this Act or printed
on the container.

**4. Display of Stock Position and Price List - Every dealer, who makes or**
offers to make a sale of any Bio-fertiliser, shall prominently display in his place
of business;
(i) The quantities of opening stock of different Bo-fertilisers
held by him on each day.

**Explanation: The actual stocks at any point of time during the day may be**
different from that of the displayed opening stock to the extent of sale and
receipt of such Bio-fertilisers up to the time of inspection during the day.

(ii) A list of prices or rates of such Bio-fertilisers fixed under
section 3 or the Maximum Retail Price printed on the
container by the manufacturer.

**5. Issue of cash or credit memorandum - Every dealer shall issue a cash or**
credit memorandum to a purchaser of bio-fertiliser in the manner prescribed.

**6. Control on distribution of Bio-fertilisers by manufacturers - The**
Government may by notification direct any manufacturer, to secure equitable
distribution and availability of bio-fertiliser to the farmers in time, to sell the
bio-fertilisers produced by any person in such quantities in such areas of the
State and within such periods as maybe specified in the notification.

**CHAPTER III**
**Registration of Manufacturers / Dealers**

**7. Dealers to be registered - No person including manufacturer/ dealer shall**
offer for sale or carry on business for selling Bio-fertilisers at any place except
under and in accordance with terms and conditions of its certificate of
registration granted to any person as prescribed under section 8 of this Act:

Provided that the Government may if it considers necessary or expedient by
notification exempt from the provisions of this section any person selling Biofertilisers to the farmers in such areas and subject to such conditions as may
be specified in that notification.


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**8. Application for Registration - (1) Every person desiring to obtain a**
Certificate of Registration under this Act for manufacturing bio-fertilisers by a
manufacturer shall make an application for registration to the Registering
Authority or the Controller in the manner prescribed.

(2) Every person desiring to obtain a Certificate of Registration under this
Act for selling Bio-fertilisers by a Dealer shall make an application for
registration to the Registering Authority together with the fee in the manner
prescribed:

Provided that where the applicant is a Government or a manufacturer, it
shall not be necessary to enclose a certificate of source along with the
application:

Provided further that where Bio-fertiliser, are obtained for sale from
different sources, a certificate of source from each such source shall be
furnished in the manner prescribed:

Provided also that a dealer, except a manufacturer shall not issue a
certificate of source to authorize another dealer for dealing in Bio-fertiliser.

**9. Grant or refusal of Certificate of Registration - The Registering Authority**
or as the case may be the Controller shall grant a Certificate of Registration
within 30 (thirty) days of the receipt of the application to any person in the
manner prescribed:

Provided that no Certificate of Registration shall be granted to a person,
(i) if his previous Certificate of Registration is under
suspension; or

(ii) if his previous Certificate of Registration has been
cancelled within a period of one year immediately preceding
the date of making the application; or

(iii) If he has been convicted of any offence under this Act or
any order made thereunder within three years immediately
preceding the date of making the application; or

(iv) if he fails to enclose with the application, a certificate of
source; or

(v) if the application is incomplete in any respect.

**10. Period of validity of Certificate of Registration - Every Certificate of**
Registration granted section 9 or as the case may be renewed under section 11,
unless suspended or cancelled shall be valid for a maximum period of 3 (three)
years from the date of issue.

**11. Renewal of certificate of Registration - (1) Every holder of a Certificate of**
registration desiring to renew the Certificate of registration granted under
section 9 shall, before the date of expiry of such certificate of registration make
an application for renewal to the Registering Authority or as the case maybe, to
the Controller as may be prescribed in duplicate, together with the fee
prescribed under section 30 of the Act for such renewal and a certificate of
source as required under section 8 of the Act.

(2) On receipt of such application together with such fee and certificate of
source, the Registering Authority, or as the case may be, the Controller may
renew the certificate of registration as may be prescribed:


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Provided that a Certificate of registration shall not be renewed if the
holder of the certificate of registration did not sell any bio-fertiliser during the
period of one year immediately preceding the date of expiry of the period of
validity of the Certificate of registration sought to be renewed.

(3) If any application for renewal is not made before the expiry of the
period of validity of the Certificate of registration but is made within one month
from the date of such expiry, the Certificate of registration may be renewed on
payment of such additional fee as may be prescribed by the Government, or as
the case may be the Controller, in addition to the fee for renewal of the
certificate of registration.

(4) Where the application for renewal is made within the time specified in
sub-section (1) or sub-section (3) the application shall be deemed to have held
a valid certificate of registration, until such date as the Registering Authority or
as the case may be the Controller passes orders on the application for renewal.

(5) If an application for renewal of a certificate of registration is not made
within one month from the date of expiry of the period of validity of the
certificate of registration, the certificate or registration shall be deemed to have
lapsed on the date on which its validity expired and any business carried on or
after that date shall be deemed to have been carried on in contravention of
section 7 of the Act.

**CHAPTER IV**
**Restriction on Manufacture, Sale etc. of Bio-Fertilisers**

**12. Restriction on Manufacture, sale and distribution of Bio-Fertilisers –**
(1) No person shall himself or by any other person on his behalf,
(i) manufacture for sale, sell, offer for sale, stock or exhibit
for sale or distribute any Bio-fertiliser which is not
prescribed standard;

(ii) manufacture for sale, sell, offer for sale, stock or exhibit
for sale, or distribute any Bio-fertiliser, which is not of
prescribed standard and which does not conform to the
particulars specified in the certificate of manufacture
granted to him under this Act in respect of Bio-fertilisers;

(iii) Sell, offer for sale, stock or exhibit for sale or distribute,
(a) any Bio-fertiliser, the container whereof is not
packed and marketed in the manner laid down in this
Act;

(b) any Bio-fertiliser which is an imitation or substitute
for another Bio-fertiliser under the name of which it is
sold;

(c) any Bio-Fertiliser which is adulterated.

**Explanation: A Bio-Fertiliser shall be deemed to be adulterated, if it contains**
any substance addition of which is likely to eliminate or decrease the living
organisms or make the fertiliser not conforming to the prescribed standard;


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(d) any Bio-fertiliser, the label or container whereof bears the name
of any individual, firm or company purporting to be manufacturer
of the Bio-fertiliser, which individual, firm or company is fictitious
or does not exist;

(e) any Bio-fertiliser, the label or container whereof or anything
accompanying therewith bears any statement which makes a false
claim for the Bio-fertiliser or which is false or misleading in any
material particular;

(f) any substance purported to be a Bio-fertiliser which substance
is not, infact, a Bio-fertiliser; or

(g) any bio-fertiliser which is not properly labelled by exhibiting the
minimum guaranteed count of living organisms.

(2) Any manufacturer who intends to manufacture for sale/offer for sale/
stock, or exhibit for sale or distribute any bio-fertiliser other than those
specified in the Schedule-I shall make an application to the prescribed
authority and the same shall be scrutinized by the Technical Committee
appointed for this purpose by the Government and be recommended to the
Government for final orders by notification by the Government in the manner
prescribed.

**13. Manufacturers to comply with certain requirements for Laboratory**
**facility - Every manufacturer shall in order to ensure the quality of their**
product possess minimum lab facility as may be specified form time to time by
the Controller.

**14. Bulk sale of Bio-fertilisers - Notwithstanding anything contained in this**
Act,
(i) a dealer shall not retain any container of any variety of
bio-fertiliser in an open and unsealed condition for the
purpose of sale;

(ii) the Government may by notification authorize a
manufacturer to sell any Bio-Fertiliser manufactured by him
in bulk also direct to the farmers for such period as may be
specified in that notification:

Provided that certificate indicating the minimum
guaranteed living cells be issued by the manufacturer to
each farmer at the time of such sale.

**15. Disposal of non-standard Bio-fertilisers - (1) Not withstanding anything**
contained in this Act, no person shall sell or offer for sale, stock, exhibit for
sale or distribute, any bio-fertiliser which does not conform to the prescribed
standards unless the Bio-fertiliser is reprocessed by the manufacturer under
the supervision of enforcing authorities, in the manner prescribed.

**16. Manufacturers to appoint officers responsible for compliance of the**
**Act - Every manufacturing organization shall in consultation with the**
Government, appoint an officer in that organisation who shall be responsible
for compliance with the provisions of this Act.


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**17. Restriction of sale/use of Bio-fertilisers - No person shall, except with**
prior permission of the Government, and subject to such terms and conditions
as may be imposed by the Government, sell or use Bio-Fertilizers for purposes
other than fertilisation of soils and increasing productivity of crops.

**CHAPTER-V**
**Enforcement Authorities**

**18. Appointment of Controller and Registering Authority- (1) The**
Government may by notification appoint an officer as the Controller, for the
purposes of this Act.

(2) The Government may by notification appoint such number of persons
as it thinks necessary, to be the registering authorities for the purposes of this
Act and may in any such notification define the limits of the local area within
which each such registering authority shall exercise his jurisdiction.

**19. Appointment of Inspectors - The Government may be notification appoint**
such number of persons, as it thinks necessary to be Inspectors of BioFertilisers for the purpose of this Act, and may in such notification define the
limits of local area within which each such inspector shall exercise his
jurisdiction.

**20. Qualification for appointment of Inspectors - No person shall be eligible**
for appointment as Bio-fertiliser Inspector, under this Act unless he possesses
the qualification namely Graduation in Agriculture from a recognized
University and working in the Department of Agriculture, in the State.

**21. Powers of Inspectors - (1) An Inspector may with a view to securing**
compliance with this Act,
(i) require any manufacturer or a dealer to give any information in
his possession, with respect to the manufacture, storage and
disposal of any Bio-fertiliser manufactured or in any manner
handled by him;

(ii) draw samples of any Bio fertilizer in accordance with the
procedure for drawal of samples laid down in Schedule II:

Provided that the Inspector shall prepare the sampling details in
duplicate as may be prescribed and hand over one copy of the
same to the dealer or his representative from whom the sample has
been drawn;

(iii) enter upon and search any premises where any Bio-fertiliser is
manufactured or stored or exhibited for sale, if he has reason to
believe that any Bio-fertiliser has been or is being manufactured,
sold, offered for sale, stored, exhibited for sale or distributed
contrary to the provisions of this Act;

(iv) seize or detain any Bio-fertiliser in respect of which he has
reason to believe that a contravention of this Act has been or is
being or is about to be committed;

(v) seize any books of accounts or documents relating to
manufacture, storage or sale of Bio-fertiliser etc in respect of which


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he has reason to believe that any contravention of this Act has
been or is being or is about to be committed:

Provided that the Inspector shall give a receipt for such Biofertilisers or books of accounts or documents so seized to the
person from whom the same has been seized:

Provided further that the books of accounts or documents so
seized shall be returned to the person from whom they were seized
after copies thereof or extracts there from as certified by such
person, have been taken:

Provided also that the Inspector shall give the stop sale
notice in writing to the person whose stocks have been detained
and initiate appropriate action as per the provisions of this Act or
the rules made thereunder within a period of twenty one days. If
no action has been initiated by the Inspector within the said period
of twenty one days from the date of issue of the said notice, the
notice of stop sale shall be deemed to have been revoked.

(2) Where any Bio-fertiliser is seized by any Inspector under this section
he shall forthwith report the fact of seizure to the respective Magistrate and
seek further orders.

**CHAPTER VI**
**Analysis of Samples**

**22. Analysis of samples - Bio-fertiliser sample drawn by an Inspector shall be**
analyzed in accordance with the standards laid down in Schedule -I as per the
specification of Bureau of Indian Standards.
**23. Qualification for appointment of Bio-fertiliser Analyst in the Bio-**
**fertiliser Control Laboratories - No person shall be eligible for appointment**
as Bio-fertiliser analyst for analysis of Bio-fertiliser samples in the laboratories
as may be notified for the purposes of this Act, unless he possesses the
qualification namely Graduation in Agriculture from a recognized University
with training in Bio-fertilisers quality control and analysis at National Centre
for Organic Farming, Ghaziabad, or its regional centres:

Provided that the Bio-fertiliser analysts appointed before the
commencement of this Act, who do not possess the requisite training, shall
undergo the prescribed training with a period of one year National Centre for
Organic Farming, Ghazibad, or its regional centres.

**24. Time limit for analysis and communication of results - (1) Where a**
sample of Bio-fertiliser has been drawn, the same shall be dispatched along
with memorandum in the manner prescribed to such laboratory as may be
notified for analysis within a period of 7 (seven) days from the date of its
drawal.

(2) The Laboratory shall analyse the sample and forward the analysis
report in the form as may be prescribed within 30 (thirty) days from the date of
receipt of the sample in the laboratory to the authorities specified in the said
memorandum.


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(3) The authority to whom the analysis report is sent under sub-section
(2) shall communicate the result of analysis to the dealer/manufacturer from
whom the sample was drawn within 15 (fifteen) days from the date of receipt of
analysis report.

(4) The report of the Laboratory is final for initiating any criminal
proceedings against the dealer/manufacturer.

**CHAPTER VII**
**Miscellaneous**

**25. Suspension cancellation of Registration Certificate - (1) A registering**
authority or, as the case maybe, the Controller may, after giving the holder of a
certificate of registration, or a certificate of manufacture or any other certificate
granted under this Act, an opportunity of being heard, suspend or cancel such
certificate on any of the following grounds namely: 
(i) that such certificate has been obtained by fraud or
misrepresentation as to material particulars:

(ii) that any of the provisions of this Act or any of the terms
and conditions of such certificate has been contravened or
not fulfilled:

Provided that while cancelling the certificate the holder
thereof may be allowed a period of 30 (thirty) days to dispose
of the balance stock of bio-fertilisers if any held by him:

Provided further that the stock of Bio-fertiliser lying
with the holder after the expiry of the said 30 (thirty) days
period shall be confiscated:

(2) Where the contravention alleged to have been committed by a person
is such as should, on being proved, justify cancelling of the certificate of
registration or, as the case may be certificate of manufacture or any other
certificate granted under this Act to such person the registering authority or as
the case may be, the Controller may without any notice, suspend such
certificate as an interim measure:

Provided that the registering authority, or as the case may be the
Controller shall immediately furnish to the affected person/persons details and
the nature of contravention alleged to have been committed by such
person/persons and, after giving the person/persons an opportunity of being
heard, pass final orders either revoking the order of suspension or cancelling
such certificate within fifteen days from the date of issue of the order of
suspension:

Provided further that where no final order is passed within the period as
specified above, the order of interim suspension shall be deemed to have been
revoked without prejudice, however to any further action which the Registering
authority or, as the case may be the Controller may take against the holder of
the certificate of registration under sub-section (1).

(3) Whenever a certificate is suspended or cancelled under this section,
the registering authority or as the case may be, the controller shall record a
brief statement of reasons for such suspension or, as the case may be


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cancellation and furnish a copy thereof to the person whose certificate has
been suspended or cancelled.

(4) Whenever a person is alleged to have committed the contravention,
the registering authority shall, within a period of fifteen days from the date of
issue of such order of suspension or cancellation furnish to the Controller also,
besides sending the same to the person whose certificate has been suspended
or cancelled, a detailed report about the nature of contravention committed
and a brief statement of the seasons for such suspension or, as the case may
be cancellation:

Provided that the Controller, shall in case of the order of suspension
passed by the registering authority, on receipt of the detailed report and after
giving the person an opportunity of being heard, pass final order either
revoking the order of suspension or cancelling the certificate of registration,
within fifteen days from the date of receipt of the detailed report from the
registering authority, failing which the order of interim suspension passed by
the registering authority shall be deemed to have been revoked, without
prejudice however to any further action which the Controller may take against
the holder of certificate of registration under subsection (1).

Provided further that the order of cancellation passed by the registering
authority shall remain effective as if it has been passed by the Controller till
such time the Controller, on receipt of the detailed report from the registering
authority and if deemed necessary after giving the person a fresh opportunity
of being heard, pass the final order either revoking or confirming the order of
cancellation.

**26. Appeal - Any person aggrieved by any of the following orders or action of**
registering Authority or as the case may be the Controller may appeal to such
authority as may be prescribed by the Government,
(i) refusing to grant, amend or renew the certificate of
registration for sale of Bio-fertilisers.

(ii) refusing to grant a certificate of manufacture for
production of Bio-fertilisers.

(iii) suspending or cancelling a certificate of registration of a
manufacturer/ dealer.

(iv) Non issuance of certificate of Registration to any person
within a period of 60 (sixty) days from the date of receipt of
such order or as the case may be from the date of expiry of
such stipulated period, and the decision of such authority
shall be final.

**27. Grant of duplicate copies of certificate of Registration etc., - Where a**
certificate of registration or a certificate of manufacture or any other certificate
granted or as the case may be, renewed under this Act is lost, torn or spoiled or
defaced as the case may be, the registering authority or as the case may be, the
Controller may on an application made in this behalf together with the fee
prescribed for the purpose under Section 30 of the Act grant a duplicate copy
of such certificate.

**28. Amendment of Certificate of Registration - The registering authority or**
as the case may be the Controller may amend the certificate of Registration on


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an application made in this behalf together with the fee as prescribed under
Section 30 of the Act.

**29. Maintenance of Records and submission of returns etc.,-(1)The**
Controller may be an order made in writing direct the dealer/ manufacturer,
(i) to maintain such books of accounts, records etc, relating
to their business in form as may be prescribed, and

(ii) to submit to such authority, returns and statements in
such form and containing such information relating to their
business and within such time as may be specified in this
Act.

(2) Where a person holds certificate of registration for sale of Bio
fertilisers, he shall maintain separate books of Account for the sales made by
him.

(3) Where the Government or a manufacturer holds a valid certificate of
registration for sale of Bio-fertilisers, they shall maintain separate books of
accounts for the sales made by them.

**30. Fees - (1) The fees payable for grant, amendment or renewal of certificate of**
registration, or certificate of manufacture, or grant of duplicate of such
certificates or renewal thereof under this Act shall be such as the Government
may from time to time fix for different purposes.

(2) The authority to whom and the manner in which the fee fixed under
sub section (1) shall be such as may be notified by the Government.

(3) Any fee paid under subsection (1) shall not be refundable unless the
grant or renewal of any certificate of registration or certificate of manufacture
to grant of duplicate copy of such certificate or renewal under this Act has been
refused.

**31. Offences and penalties - (1) Whoever, -**

(i) manufactures, sells, stocks or exhibits for sale or
distributes any Bio-fertiliser deemed to be nonstandard
under Section 2 (10); or

(ii) manufactures, sells, stocks or exhibits for sale or
distributes a Bio- fertiliser without a certificate of
Registration; or

(iii) manufactures, sells or distributes a Bio-fertiliser in
contravention of Section 12; or

(iv) obstructs a Bio-fertiliser inspector in the exercise of his
powers of charge of this Act,
shall be punishable,
(a) for the first offence, with imprisonment for a term which may
extend to one year or with fine which may extend to rupees fifty
thousand, or with both;

(b) for the second and subsequent offence with imprisonment of a
term which may extend to two years or with fine which may extend
to rupees one lakh, or with both.


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(2) Whoever contravenes any other provisions of this Act or any
conditions of Certificate of registration granted thereunder shall be
punishable;
(a) for the first offence with imprisonment which may extend to six
months or with fine which may extend to five thousand rupees or
with both;

(b) for the second subsequent offence with imprisonment for a term
which may extend to two years, or with fine which may extend to
twenty thousand or with both.

(3) If any person convicted of an offence under this Act committees a like
offence afterwards, it shall be lawful for the Court before which the second or
subsequent conviction takes place to cause the offenders name and place of
residence, the offence and penalty imposed to be published in such
newspapers or in such other manner as the court may direct.

**32. Protection of action taken in good faith - No suit, prosecution or other**
legal proceedings shall lie against the Government or any officer, authority or
person empowered to exercise the powers and perform the functions by or
under this Act for anything which is done in good faith or intended to be done
under this Act or the rules or orders made thereunder.

**33. Power to remove difficulties - If any doubt or difficulty arises in giving**
effect to the provisions of this Act, the Government may, by order make such
provisions or give such directions not inconsistent with the provisions of this
Act as may appear to it to be necessary or expedient for removal of doubt or
difficulty:

**34. Power to make Rules - (1) The Government may, by notification, make**
rules for carrying out all or any of the purpose of this Act.

(2) Every rule made under this Act shall immediately after it is made, be
laid before the Legislative Assembly of the State, if it is in session and if it is
not in session, in the session immediately following for a total period of
fourteen days which may be comprised in one session or in two successive
sessions, and if, before the expiration of the session in which it is so laid or the
session immediately following the Legislative Assembly agrees in making any
modification in the rule or in the annulment of the rule, the rule shall, from the
date on which the modification or annulment is notified, have effect only in
such modified form or shall stand annulled as the case may be, so however,
that any such modification or annulment shall be without prejudice to the
validity of anything previously done under that rule.

**35. Repeal of Ordinance 23 of 2005 - The Andhra Pradesh Bio-Fertilisers**
(Monitoring and Quality Control) Ordinance, 2005 is hereby repealed.

**SCHEDULE-I**

[See Section 2(1)]
**PART-A**
**SPECIFICATIONS OF BIO - FERTILISERS**

**1. NITROGEN FIXING BIO-FERTILISERS: -**

**a. RHIZOBIUM INOCULANTS (RI) (IS 8268 - 2001)**


-----

(i) RI shall be carried based, the colour depending on the
colour of the carrier

(ii) RI shall contain a minimum of 10[7] 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 microorganisms at 10[5] dilution when counted as given in 4.3 of 1
A 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 stray 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 and (NBDC),**
Ghaziabad and its Six Regional Centres located at Bangalore, Bhubaneshwar,
Imphal, Hissar, Jabalpur 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.

**(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 10[7] viable Azotobacter
cell/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


-----

Para B of Schedule II and Azotobactor Chroococcum 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 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 10[5] 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 14806-2000)**

(i) ASI shall contain 10[7] viable Azospirillum cells / g of the
carrier material on dry mass basis.

(ii) ASI shall have no contamination with other
microorganisms at 10[5] dilution. ASI 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 ASI carrier shall be in the form of fribal (moist) with
30-40 percent moisture. When tested as given in Annexure-B

(v) ASI shall show effective root development on all
cultivars/crops against which the Inoculant is intended to be
used.

(vi) Specified mother culture be obtained from any institution
maintaining the mother cultures. The manufacturer may
control the quality of the both, 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) PSIB shall contain 10[7] 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 sterilised. When tested it shall pass
through 100 micron sieve.

(iii) PSBI shall have no contamination with other
microorganisms at 10[5] 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.

**SCHEDULE-I**
**PART B**
**PACKING MARKING AND STORAGE**

**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 inoculant

(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 colour 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 in governed by the provisions of the
Bureau of Indian Standards Act, 1986 and the rules and regulations made
there under. The details of conditions under which the license 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 15[0] C to 30[0] C. It shall also be
the duty of the manufacture to instruct the retailers and, in turn the users
about the precautions to be taken during storage.

**2. AZOTOBACTOR:**

**1. Packing - AI shall be packed in packaging material of low density polythene**
/ polypropylene bags thickness of which shall be 75-100 micron minimum.

**2. Marking - 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
there under. The details of conditions under which the license 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 15°C to 30°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.

**3. AZOSPIRILLIUM:**

**1. Packing - ASI shall be packed in polyethylene packs, thickness which shall**
not be less than 75-100 micron.

**2. Marking - Each polyethylene packs shall be marked legibility and indelibly**
with the following information.

(a) Name of the product, specially as Azospirillum 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 preferable 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 there under.
The details of conditions under which the license 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 20°C and not exceeding 30°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.

**4. PHOSPHATES SOLUBLISING BACTERIAL INOCULANT (PSBI)**

**1. Packing - PSBI shall be packed in polyethylene packs, thickness of which**
shall not be less than 100 micron.

**2. Marking - Each polyethylene packs shall be marked legibly and indelibly**
with the following information.


-----

(a) Name of the product, specially as Phosphate solubilising
bacterial 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 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 there under.
The details of conditions under which the license 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 cool and dry place away
from direct heat preferably at a temperature of 20°C and not exceeding 30°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.

**SCHEDULE-II**
**PART-A**

**[See Section 21]**
**PROCEDURE FOR SAMPLING OF BIO-FERTILISERS**

**1. General Requirements**
1.0. In drawing, preparing and handling the samples, following
precautions and direction 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 preservation or bactericidal/ fungicidal agent shall be added to
samples required for micro-biological 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 conditions 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 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. Sample 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 sterilised by heating in a hot air oven at 160°C for not less than 2h
or by autoclaving for not less than 20 min at 120°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. Drawl 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 From K along with other details like sample no. / code no. which
enables its identification.

**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[0] 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

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. 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[10] Take
0.2 ml or suitable aliquots of 10[6] to 10[9] 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[8] 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

Agar 20 g

Sucrose (C12 H2 Ol1) 20.0 g

Ferric sulphate (Fe2 (S04)2) 0.1 g

Dibasic potassium phosphate 1.0 g
(K2HP04)

Magnesium sulphate 0.5 g
(MgS04, 7H20)

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[10] Take
0.2 ml or suitable aliquots of 10[6] to 10[9] 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[0] 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 acid 5.0 g

Potassium hydroxide 4.0 g

Di-potassium hydrogen 0.5 g
phosphate

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 1000ml


Boromothymol blue

(0.5% alcoholic solution)


2.0 m.


Agar 1.7 g

pH adjusted to 6.5 - 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.

**MPN table value x Dilution level**
**Azospirillum Count/g of carrier =**
**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:

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. 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[7]

(c) Take 0.2 ml aliquote of 10[5] to 10[7] 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.

Mannitol 10.0 g

Potassium hydrogen phosphate0.5 g

(k2HP04)

Magnesium sulphate (MgS04 0.2 g
7H20)

Sodium chloride (NaCl) 0.1 g

Calcium Carbonate (Ca C03) 1.0 g

Yeast extract 1.0 g

Agar 18.0 g

Distilled water 1 liter

pH 6-8-7.0

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.


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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

Iodine 1.00 g

Potassium 2.00 g
Iodide

Distilled 300 ml
water

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

c. Erythrosine

Erythrosine 1.00 g

Phenol 5.00 g

Distilled 100 ml
water

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.


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**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:

Glucose 10.0 g

Peptone 20.0 g

Sodium chloride (NaCI) 5.0

Agar (IS 6850} 15.0

Distilled water 1000 ml

Bromocresol purple 10 ml of

ethyl alcohol solution 1.6 persons

pH 7.2

**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
10[6] to 10[9]. 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[8] to 10[9] 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

Agar 20 gm

Sucrose 20 gm

Ferric Sulphate 0.1


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Dibasic Potassium Phosphate 1.0 gm

Magnesium Sulphate 0.5 gm

Calcium carbonate 2.0 gm

Sodium Molybdate 0.005 gm

Distilliled water 1000 ml

PH 6.8 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


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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[8] to 10[9] 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


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**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.**


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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.


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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.


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