ICS 65.080  
 
Reference number  
DRS 488 -1:2022  
© RSB 2022  
  
 RWANDA 
STANDARD  WD 
488-1 
 
First edition  
2022 -mm-dd 
 
Bio-inoculants  — Specification  — 
Part 1: Nitrogen fixing bacteria  
   
DRS 488 -1: 2022  
©RSB 2022  - All rights reserved                  ii 
 In order to match with technological development and to keep continuous progress in industries, 
standards are subject to periodic review. Users shall ascertain that they are in possession of the latest 
edition  
 
 
 
 
 
 
 
 
© RSB 2022 
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized 
in any form or by any means, electronic or mechanical, including photocopying and microfilm, without 
prior written permission from R SB. 
Requests for permission to reproduce this document should be addressed to : 
Rwanda Standards  Board  
P.O Box 7099 Kigali -Rwanda  
KK 15 Rd, 49  
Tel. +250 788303492  
Toll Free: 3250  
E-mail: info@rsb.gov.rw  
Website: www.rsb.gov.rw  
ePortal: www.portal.rsb.gov.rw   DRS 488 -1: 2022  
iii                ©RSB 2022  - All rights reserved  
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ ....................  v 
1 Scope  ................................ ................................ ................................ ................................ .............  1 
2 Normative references  ................................ ................................ ................................ ...................  1 
3 Terms and definitions  ................................ ................................ ................................ ..................  1 
4 Requirements  ................................ ................................ ................................ ................................  3 
4.1 General requirements  ................................ ................................ ................................ ..................  3 
4.2 Specific requirements  ................................ ................................ ................................ ..................  3 
4.2.1  Rhizobium inoculants  ................................ ................................ ................................ ..................  3 
4.2.2  Azobacter inoculants  ................................ ................................ ................................ ...................  3 
4.2.3  Azospirillum inoculants  ................................ ................................ ................................ ...............  4 
5 Packaging  ................................ ................................ ................................ ................................ ...... 5 
6 Labelling  ................................ ................................ ................................ ................................ ........  5 
7 Sampling  ................................ ................................ ................................ ................................ ....... 5 
Annex  A (normative)  Sam pling  ................................ ................................ ................................ ...............  6 
A.1 General requirements  ................................ ................................ ................................ ..................  6 
A.2 Sampling equipment  ................................ ................................ ................................ ....................  6 
A.3 Scale of sampling  ................................ ................................ ................................ .........................  7 
A.4 Criteria  of conformity  ................................ ................................ ................................ ...................  7 
A.4.1  Rhizobium  ................................ ................................ ................................ ................................ ..... 7 
A.4.2  Azotobacter  ................................ ................................ ................................ ................................ ... 7 
A.4.3  Azospirillum  ................................ ................................ ................................ ................................ .. 8 
Annex  B (normative)  Determination of pH and moisture content  ................................ .......................  9 
B.1 Determination of pH  ................................ ................................ ................................ .....................  9 
B.1.1  Solid microbial bio -fertilizers  ................................ ................................ ................................ ...... 9 
B.1.2  Liquid microbial bio -fertilizers  ................................ ................................ ................................ .... 9 
B.2 Moisture content  ................................ ................................ ................................ ...........................  9 
B.2.1  Procedure  ................................ ................................ ................................ ................................ ...... 9 
B.2.2  Calculation  ................................ ................................ ................................ ................................ .... 9 
Annex  C (normati ve)  Determination of number of Rhizobium cells  ................................ .................  10 
C.1 Reagents  ................................ ................................ ................................ ................................ ..... 10 
C.2 Preparation of serial dilution for plate count  ................................ ................................ ..........  10 
C.3 Incubation of plates  ................................ ................................ ................................ ...................  10 
C.4 Colo ny counting aids  ................................ ................................ ................................ .................  11 
C.5 Colony count  ................................ ................................ ................................ ..............................  11 
Annex  D (normative)  Test for nodulation (Pot culture test)  ................................ ...............................  12 
D.1 Plant nutrient solution  ................................ ................................ ................................ ...............  12 
D.1.1  Reagents  ................................ ................................ ................................ ................................ ..... 12 
D.1.2  Preparation of plant nutrient solution  ................................ ................................ ......................  12 
D.2 Procedure for nodulation ................................ ................................ ................................ ...........  13 
Annex  E (normative)  Total plate count of Azotobacter  ................................ ................................ ...... 15 
E.1 Preparation of culture Medium  ................................ ................................ ................................ . 15 
E.2 Preparation for serial dilution for plate count  ................................ ................................ .........  15 DRS 488 -1: 2022  
©RSB 2022  - All rights reserved                  iv 
 E.3 Incubation of plates  ................................ ................................ ................................ ...................  15 
E.4 Colo ny counting aids  ................................ ................................ ................................ ................  15 
E.5 Colony count  ................................ ................................ ................................ ..............................  16 
Annex  F (normative)  Test for nitrogen fixation in pure culture  ................................ ........................  17 
F.1 Preparation of pure culture medium  ................................ ................................ ........................  17 
F.2 Procedure  ................................ ................................ ................................ ................................ ... 17 
F.3 Determination by Kjeldahl method  ................................ ................................ ..........................  17 
F.3.1  Reagents  ................................ ................................ ................................ ................................ ..... 17 
F.3.2  Apparatus  ................................ ................................ ................................ ................................ ... 18 
F.3.3  Procedure  ................................ ................................ ................................ ................................ ... 18 
F.3.4  Calculation  ................................ ................................ ................................ ................................ .. 19 
F.4 Determination of sucrose consumed  ................................ ................................ ......................  19 
F.4.1  Sample preparation  ................................ ................................ ................................ ...................  19 
F.4.2  Method of inversion  ................................ ................................ ................................ ...................  19 
F.4.3  Determination of reducing sugars  ................................ ................................ ...........................  19 
F.5 Calculation  ................................ ................................ ................................ ................................ .. 21 
F.5.1  Total reducing sugars  ................................ ................................ ................................ ...............  21 
F.5.2  Determination of sucrose  ................................ ................................ ................................ .........  21 
F.5.3  Determination of nitrogen fixed per g of sucrose consumed  ................................ ...............  21 
Annex  G (normative)  Determination of the number of Azospirillum cells  ................................ ....... 22 
G.1 Medium  ................................ ................................ ................................ ................................ ....... 22 
G.2 Preparation of serial dilution for MPN count  ................................ ................................ ..........  22 
G.3 Counting  ................................ ................................ ................................ ................................ ..... 22 
Annex  H (normative)  Test for effective root growth  ................................ ................................ ...........  23 
H.1 Pot Culture Test  ................................ ................................ ................................ .........................  23 
H.2 Method f or estimating MPN count for Azospirillum  ................................ ...............................  23 
H.3 Calculation  ................................ ................................ ................................ ................................ .. 24 
Annex  I (normative)  Guidelines on maintenance and preparation of culture and quality control at 
broth stage  ................................ ................................ ................................ ................................ . 25 
I.1 Rhizobium  ................................ ................................ ................................ ................................ ... 25 
I.1.1  Maintenance of pure cultures  ................................ ................................ ................................ ... 25 
I.1.2  Preparation of inoculum cultures  ................................ ................................ ............................  25 
I.1.3  Preparation of mass culture  ................................ ................................ ................................ ..... 25 
I.1.4  Quality control tests recommended at broth stage  ................................ ...............................  26 
I.2 Azospirillum  ................................ ................................ ................................ ...............................  27 
I.2.1  Maintenance of pure cultures  ................................ ................................ ................................ ... 27 
I.2.2  Preparation of inoculums culture and mass culture ................................ ..............................  28 
I.2.3  Quality control test recommended at broth stage  ................................ ................................ . 28 
I.3 Azotobactor  ................................ ................................ ................................ ................................  28 
I.3.1  Maintenance  ................................ ................................ ................................ ...............................  28 
I.3.2  Preparation of inoculums culture  ................................ ................................ ............................  29 
I.3.3  Quality control tests recommended at broth stage  ................................ ...............................  29 
 DRS 488 -1: 2022  
v                ©RSB 2022  - All rights reserved  
 Foreword   
Rwanda Standard s are prepared  by Technical Committees and approved by Rwanda Standards Board (RSB) 
Board  of Directors in  accordance with the procedures of RSB, in compliance with Annex 3 of the WTO/TBT 
agreement on the preparation, adoption and application of standards.  
The main task of technical committees is to prepare national st andards.  Final Draft Rwanda S tandards adopted 
by Technical committ ees are ratified  by me mbers of RSB Board of Director s for publication and gazettment as 
Rwanda Standards.  
DRS 488 -1 was prepared by Technical Committee RSB/TC 007, Agrochemicals . 
In the preparation of this standard, reference was made to the following standard:  
WD-ARS 1452 -1:2017 , Microbial bio -fertilizers  — Part 1: Specifications for nitrogen fixing bacteria  
The assistance de rived from the above source is hereby acknowledged with thanks.  
DRS  488 consists of the following parts, under the general title Bio-inoculants — Specification : 
⎯ Part 1: Nitrogen fixing bacteria  
⎯ Part 2: Phosphate solubilizing bacteria  
⎯ Part 3: Potash solubilizing bacteria  
Committee membership  
The following organizations were represented on the Technical Committee on Agrochemicals  (RSB/TC 007) in 
the preparation of this standard.  
AGROPY Ltd  
University of Rwanda/College of Education (UR/CE)  
Ministry of Environment (MoE)  
University of Rwanda/Colle ge of Sciences and Technology (UR/CST)  
Rwanda Inspectorate, Competition and Consumer Protection Authority (RICA)  
Rwanda Forensic Laboratory (RFL)  
Rwanda Agriculture and Animal Resources Development Board (RAB)  DRS 488 -1: 2022  
©RSB 2022  - All rights reserved                  vi 
 National Agricultural Export Development Board  (NAEB)  
Rwanda Investigation Bureau (RIB)  
Rwanda  Standards Board  (RSB) – Secretariat  DRS 488 -1: 2022  
vii                ©RSB 2022  - All rights reserved  
 Introduction   
Commercial farming to meet food security involving intensive  land use relies heavily on agricultural inputs 
such as chemi cal fer tilizers and synthetic pesticides. Ho wever, increasing continuo us use of chemical 
fertilizers over yea rs has caused environmental d amage such as groundwater contamination, eutrophication  
of water  bodies. Lo ng term sole reliance  on excess ive use of chemical fertilizers and synthetic pes ticides has 
also adve rsely im pacted on soil quality, fertility and soil microbial biodivers ity. 
With the increasing demand for food to meet the gro wing population, food produc tion can be enhanced by 
increasing the pr oductivity of existing agricultural land sustainably and/or bring additional land under cultivation. 
Ensuring sustainable long term land productivity can  be achieved through an integrated nutrient management 
system inv olving a c ombination of environmental ly friendly sources  of plant nutrients including organic 
microbial fertilizers and chemical fertilizers. 
Bio-inoculant s are  defined as preparations conta ining living cells or latent  cells of efficient strains of 
microorg anisms that help crop plants’ uptake of nutrients by their interac tions in the rhizosphere when 
applied throu gh seed or soil. They accelerate certain microbial processes in the soil which augment the 
extent  of availability of nutrients in a form easily assimilated by plant s. Bio-inoculant s gene rate plant nutrients 
like nitrogen and phosphorus thr ough their a ctivities in the s oil or rhizosphere and make available in a gradual  
manner to plants.  
Types  of bio‐inoculant s 
a) Nitrogen  fixers  
1) Bacteria – eg. Rhizobium, Bacillus, Mycobacterium, Azotobacter and Azospirillum 
2) Blue green algae – eg. Anabaena, Nostac, Tolypo tlurix, 
b) Phosphate  solubilising – eg. Pseudomon as spp. 
c) Plant  growth promoting  Rhizobac teria 
d) Sulphur  solubilising microbes 
e) Azolla – eg. Azolla filicoloides, Azolla rubra 
f) Vesicular  Arbuscular Mycorrhiza (VAM fungi) 
Potential benefits   
Potential benefits of using bio‐inoculant s are: 
a) To provi de an eco‐friendly alternat e renewable  source of nutrients.  
b) Sustain  soil health  DRS 488 -1: 2022  
©RSB 2022  - All rights reserved                  viii 
 c) Decompose plant residues, and stabilize C:N ratio of soil  
d) To enhance  soil biolog ical activity and improve availability of plant nutrients.  
e) To improve  chemical, physica l and biolo gical properties of soil. 
f) To promote  plant growth and improve crop yield.  
g) Solubilize and mobilize nutrients  
Bio-inoculants  are supplement  to chemical  fertilizers but not substitute to it. They usually cause 20 to 30 
percent  increase  in crop yield. Ho wever, they do not cause  marked increase in produc tivity like chemicals. 
Efficiency of bio-inoculants  is marked ly dep endent on soil charac ter, for example moisture content, pH, 
tempera ture organic  matter and types  of resident  microorganism. When  these factors are unfavourable 
microbial fertilizers  may not be effective in increasing the soil fertility. DRS 488 -1: 2022 
1                ©RSB 2022  - All rights reserved  
  Bio-inoculants  — Specification  — Part 1: Nitrogen fixing bacteria  
1 Scope   
This Draft  Rwanda Standard  specifies the requirements and method s of test for three types of nitrogen  fixing 
microbia l bio‐fertilizers:  
(i) Rhizobium sp  
(ii) Azotobacter sp  
(iii) Azospirillu m spp 
This standard is applicable to both solid as well as liquid nitrogen  fixing bio‐inoculants .  
This part of the standard  is not applicable to other types  of bio‐fertilizers such as algae, ferns, fungi an d others.  
2 Normative references   
The following documents are referred to in the text in such a way that some or all of  their content constitutes 
requirements of this document.  For dated references, only the edition  cited applies. For undated references, the 
latest edition of t he referenced document (including any amendments) applies.  
RS ISO 8157 , Fertilizers and Soil conditioners — Vocabulary  
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and perfo rmance 
testing  of culture media 
3 Terms and definitions   
For the purposes of this standard , the terms and definitions given in RS ISO 8157 and the following apply . 
3.1 
Azosprillum inoculant  (ASI) 
a product having large population of a strain or a combination of seve ral strains of Azosp irillum for particular 
graminaceous plants  which develop efficient roots in the plant and enrich soil with nitrogen  and thereby 
enhance nitrogen fixation, crop growth  and productivity  
3.2 
Azotobact er inoculants (AI)  
©RSB 2022  - All rights reserved                  2 
 a product  having a large population of  a strain or a combination of  several strains of Azobacter for a 
particular group of non‐leguminous plants  
3.3  
inoculants 
these are preparations  containing beneficial micro‐organi sms in a viable  state, intended for seed or soil 
application, designed to impr ove soil fertility and help plant growth by inc reasing the number of desired 
microorganism  in plant root zone  
3.4 
bio‐inoculant  
product containing carrier  based (solid or liquid) living microorganisms w hich are agriculturally useful in 
terms of nitrogen fixation, p hosphorus solubili zation or nutrient mobilization when applied to seed, plant 
surfaces, or soil colonizes the rhiz osphere or int erior of plants and prom otes growth by increasing the supply or 
availability of primary nutrients to ho st plants through nat ural processes like Nitroge n fixation, Solu bilizing 
phosphorus and stimulating plant growth through  the synthesis of growth  promo ting substances to increase 
the productivity of the soil and/or  crop. 
3.5 
bio‐inoculant  carrier 
substance which act as a carrier material  to support and keep organisms  in a viable state. 
NOTE The carrier material such as peat, lignite, peat soil, humus, wood charcoal or similar material favouring growth of the organ ism. 
3.6 
Rhizobium inoculants (RI) 
a product having a large population of a strain or a combination of seve ral strains of Rhizobium sp colonizing 
the roots  of differ ent leguminous plants  and reducing atmospheric nitrogen symbiotically to  ammo nia wh ich 
is then utilized by the plant to produce root  nodules thereby enhancing nitrogen fixation, crop grow th and 
yield. 
3.7 
lot 
all units in a single consignment of type of material  belonging to the same batch of manufacture 
 DRS 488 -1: 2022 
3                ©RSB 2022  - All rights reserved  
  
4 Requirements  
4.1 General requirements  
4.1.1  For solid bio‐inoculant s, the carrie r used shall be of a nature that is not harmfu l to the environm ent and 
the colour of the  inoculants  would be depend ent on the colour of the carrie r. Peat soil, lignite, vermiculite, 
charcoal, press mud, farmyard manure and soil mix ture can be used as carrier  materials.  
4.1.2 The physical features  of liquid rhizobium bio-inoculant s shall be :  
a) dull white in colour,  
b) no bad smell,  and  
c) no foam formation  
4.2 Specific requirements  
4.2.1  Rhizobium inoculants  
The rhizobium  bio‐inoculant ss shall comply with the requiremen ts specified in Table 1. 
Table 1 — Requ iremen ts for Rhizobium  bio‐fertilizers 
 Parameter  Requirement  Methods of test 
(1) Viable  count, CFU,  min 5.0 x 107 cell/solid carrier or 
1.0 x108 cell/ml  of liquid Annex  C 
(2) Contamination  level No contam ination  at 10‐5 dilution  Annex  C 
(3) pH 6.5 ‐ 7.5 Annex  B 
(4) Partic les size distribution, 
(in case  of carrier based)  All materials pass 0.15 ‐ 0.212 mm sieve   
(5) Moisture content, %, max 
(in case  of carrier based)  30 ‐ 40  Annex  B 
(6) Efficiency  character Shall show effecti ve nodulation on all the 
species l isted on the label.  Annex D 
4.2.2  Azobacter inoculants  
4.2.2.1  The physical feature of liquid Azotobacter bio‐inoculant s: The pigmentation that is produced by 
Azotobac ter in aged  cultu re is melanin. Azotobacter . chroococcum produces  brown‐black pigmentation in 
liquid inoculum. 
4.2.2.2  The Azotobacter bio‐inoculant s shall comply  with the requiremen ts specified in Table  2  
©RSB 2022  - All rights reserved                  4 
 Table  2 — Requirements  for Azotobacter bio‐inoculants  
 Parameter Requi remen ts Methods  of test 
(1) Viable cell cou nt, 
CFU,  min 5.0 x 107 cell/ solid carrier  
or 
1.0 x108 cell/ml  of liquid Annex E 
(2) pH 6.5 ‐ 7.5  Annex  B 
(3) Contamination  level No contam ination  at 10-5 dilution Annex E 
(4) Particles  size distribution, 
(in case  of carrier  based) All material shall pass through 0.15‐
0.212 mm sieve  
(5) Moisture content, % by weight, 
max (in case  of carrier  based) 30‐40 Annex  B 
(6) Efficiency  character  The strain shall be capable  of fix ing at 
least 10 mg of nitrogen per gram of 
sucrose  consumed. Annex F 
4.2.3  Azospirillum inoculants  
4.2.3.1  The physical features  of liquid Azospirillum shall comply with the following:  
d) The colour of the liquid may be blue or dull white. 
e) Bad odour confirms improper  liquid formulation and may be concluded  as mere broth. 
f) Production of yellow gummy colour materials confirms the quality produc t. 
g) Acidic pH always confirms that there are no Azospirillum bacte ria in the liquid. 
4.2.3.2  The Azospirillum bio‐inoculants  shall comp ly with the requireme nts specified in Table 3. 
Table 3 — Requ iremen ts for Azospirillum bio‐inoculants  
 Parameter Requi rement  Methods  of test 
(a) Viable  cell count, CFU,  min 5.0 x 107 cell/g  solid carrier   
or 
1.0 x108 cell/ml  of liquid Annex  G 
(b) Contamination  level No contam ination  at 10-5 dilution Annex  G 
(c) pH 6.5 – 7.5 Annex  B 
(d) Particles  size distribution, 
(in case  of carrier  based) All material shall pass through 0.15‐
0.212 mm sieve  
(e) Moisture content, %  by 
weight, max (in case  of 
carrier  based) 30 ‐ 40 Annex  B 
(f) Efficiency  character  Formation of white  pellicle in semi 
solid nitrogen  free bromothymol  blue 
media. 
 
Shall show effecti ve root developm ent 
on all cultivar/crops  Annex  H 
 DRS 488 -1: 2022 
5                ©RSB 2022  - All rights reserved  
 5 Packaging  
The bio‐inoculants  shall be packed in polyethylene packs, thickness  of which shall not be less than 100 
micron. 
6 Labelling  
Each  pack shall be marked legibly and indel ibly with the following information: 
a) Name  of product; 
b) Name,  address and phone number  of the manuf acturer; 
c) intended  crop;  
d) Type  of carrier used; 
e) Batch  number; 
f) Production  date and expiry date; 
g) Net quantity in kg or ml, 
h) Rate  of application per ha or m2; 
i) Storage instruction;  
j) Direction  for use of bio‐fertilizers shall be printed  briefly on the packet.  A separate pamphlet may 
preferably be given with it. 
k) Microbial count  (cfu/ml or cfu/g ) 
7 Sampling  
The method of d rawing representative samples of inoculants f rom different bat ches s hall be as per Annex A.  
  
©RSB 2022  - All rights reserved                  6 
 Annex  A 
(normative)  
 
Sampling  
A.1 General  requirements  
A.1.1  In drawing, preparing and handling the samples, the following precautions and directions shall be 
observed.  
A.1.2 Sampl ing shall be carried out by a trained  and experienced pe rson as it is essential that the 
sample should be representative of the lot to be examined.  
A.1.3 Since  the samples are also required  for microbiological analysis, utmost care is neces sary to avoid 
extraneo us contamination while drawing and handling the samples and to preser ve them in their original 
condit ion till they are ready for examination in the laboratory. 
A.1.4 No preservative or  bactericidal/fungi cidal agent shall be added  to samples requi red for 
microbiological analysis. 
A.1.5 Samples  in their original unopened packets should  be drawn and sent to the laborat ory. This will 
prevent possible conta mination  of the samples during handling and also help in reveal ing the true condition 
of the material. 
A.1.6 Intact packe ts shall be drawn from a prote cted place not expo sed to dampness, air, bright light, dust 
or soot, and trans ferred to clean conta iners. 
A.1.7 The sampling applian ces and sample containers shall be clean and sterile. 
A.1.8 All precautions shall be taken to prote ct the sample the material being sampled, the sampling 
instruments and the sample container against adventitious c ontamination at the time of drawing the sample, 
opening  containers and transferring the samples.  
A.2 Sampling equipment  
A.2.1  A suitable scoo p made of st ainless steel may be used for drawing samples for microbiological and 
modulation studies. 
A.2.2 The sampling equipment shall be perfectly clean and sterile. It shall be properly sterilized by heating 
in a hot air‐oven  at 160 °C for not less than 2 h or by autoclaving  for not less than 15 min at 121 C and held 
in suitable containers  to prevent  re‐contamination. 
 DRS 488 -1: 2022 
7                ©RSB 2022  - All rights reserved  
 A.3 Scale of sampling  
A.3.1  For ascertaining conformity of the mater ial to the requirements of the specification, samples shall be 
tested from each lot separately. 
A.3.2  The number  of packe ts to be selected  from a lot shall depend on the size of the lot and shall be 
according to Table A.1 or A.2 
Table  A.1 — Scale of sampling for Rhizobi um 
No of packets in the lot Sample size for 
Nodu lation Sample size for 
other parameters Acceptan ce No 
100 to 1000  2 5 0 
1001 to 5000 3 8 0 
5001 and above 4 13 1 
Table A.2 — Scale  of sampling for Azotobacter 
 Sample size 
Nitrog en Fixation Other  para meters  
Up to 100 1 6 
101 to 1000  2 9 
1001 to 10 000 3 12 
10 001 and above 4 15 
A.4 Criteria of conformity  
A.4.1  Rhizobium  
A.4.1.1  Each packet sel ected from the lot according to  column 1  and 3 of Table A.1 shall be tested for the 
requirements in Table 1,  excluding effective nodulation test. A packet failing in one or more  of these 
requiremen ts shall be considered  as defective. 
A.4.1.2  The lot, having found to be satisfact ory according to A.4.1  (i), shall be tested  for effective nodulation 
as given  in Table 1 (f). For  this test the number of packets  shall be selected from the lot according to 
column 4 of Table A.1. 
A.4.1.3  The lot shall be declared as conforming  to the requirements of the specification if none of the 
packets,  taken in accordance to column 4 in the sample fails in effective  nodulation. 
A.4.2  Azotobacter  
The lot shall be declared as conforming to the requi rements of the specification if none  of the packet 
selected in accordance  to Table A.2 fails the requirements  of specified in Table  2.  
©RSB 2022  - All rights reserved                  8 
 A.4.3  Azospirillum  
Same  as for rhizobium. 
 
  DRS 488 -1: 2022 
9                ©RSB 2022  - All rights reserved  
 Annex  B 
(normative)  
 
Determination of pH and moisture content  
B.1 Determination of pH  
B.1.1  Solid microbial bio -fertilizers  
B.1.1.1  Make 25 g of inoculants into a suspension in 50 ml of distilled water and shake on a rotary shaker 
for 2 h, filter on Whatman  No.1 filter paper or equivalent  under vacuum using a Buchner funnel.  
B.1.1.2  Determ ine pH of filtrate using a pH meter.  
B.1.2  Liquid microbial bio -fertilizers  
Take 100 ml of inoculants into a 250 ml clean  and dry conical flask and shake on a rotary  shaker for 2 h. 
Determine  pH using a pH meter. 
B.2 Moisture content  
The moisture content test is applicable only for powder granules or carrier material. 
B.2.1  Procedure  
B.2.1.1  Take the weight of an empty container  
B.2.1.2  Measure a known quantity of carrier based microbial bio -fertilizer in a pre -weighed container  
B.2.1.3  Heat the sample for 24 hours in an air oven at 80 C. 
B.2.1.4  Allow to cool in a desicca tor and take the weight. 
B.2.1.5  Repeat the process of drying to constant weight. 
B.2.2  Calculation  
Moisture content (%) = 
100sample oven the of Weightdrying on  weightin Loss   
©RSB 2022  - All rights reserved                  10 
 Annex  C 
(normative)  
 
Determination of number of Rhizobium cells  
C.1 Reagents  
C.1.1  Congo Red  – 1% of aqueous solution  
C.1.2  Preparation of culture medium : Yeast extract mannitol broth  
a) Use a plating medium of the following composition: 
Agar  20 g 
Yeast extract  1 g 
Mannitol  10 g 
Potassium hydrogen phosphate (K 2HPO 4) 0.5 g  
Magnesium sulphate (MgSO 4.7H 2O) 0.2 g  
Sodium Chloride (NaCl)  0.1 g  
Congo red  2.5 ml  
Distilled water  1000 ml  
pH 7.0 
b) The culture medium shall be prepared in accordance with ISO 11133 or using  any other acceptable 
validated test method.  
C.2 Preparation of serial dilution for plate count  
C.2.1  Dispense 30 g of Inoculants to 300 ml of sterile distilled  or demineralized wa ter and shake for 10 
min on a recipr ocal shaker or homogen izer. 
C.2.2  Make serial dilutions up to 10-10 by suspen ding 10 ml aliquot of previous dilution to 90 ml of water.  
C.2.3  Take 0.2 ml or suitable aliquot of 10-6 to 10-8 dilutions using sterile pipettes and deliver to petri 
dishes containing set  medium as given in C.1.2 and spread it uniformly with a spreader.  Invert the plates 
and promptly  place them in the incubator.  
C.3 Incubation of plates  
Label the plates and incubate at 28 ± 2 C for 3 to 5 days for fast growing Rhizobia and 5 to 10 days for 
slow‐growing  ones. DRS 488 -1: 2022 
11                ©RSB 2022  - All rights reserved  
 C.4 Colony counting aids  
Count the coloni es with the aid of magnifying lens under uniform and properly controlled, artificial illumination. 
Use a colony counter,  equip ped with a guide plate and rules in Centimeter Square.  Record the total number  
of coloni es with the hand tally. Avo id mistaking particles of undissolved me dium or precip itated ma tter, in 
plates for pinpoint colonies. To distinguish colonies from dirt, specks and other foreign matter,  examine 
doubtful objects carefully. 
C.5 Colony count  
C.5.1  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 a bsorb Congo red and stand out as reddish colonies. 
C.5.2  Rhizobium forms white, translucen t, glistening, elevated and comparatively small colonies on this 
medium. Moreover, Rhizobium colonies do not take up the colour of congo red dye added  in the medium. 
Those colonies which readily  take up the congo red stain are not rhizo bia. 
C.5.3  Count such colony  numbers and calculate  figures in terms  of per gram,  of carrier. Also check for 
freedom  from contamination at 10-8 dilution.  
©RSB 2022  - All rights reserved                  12 
 Annex  D 
(normative)  
 
Test for nodulation (Pot culture test)  
D.1 Plant nutrient solution  
D.1.1  Reagents  
 Com position Concen tration g/l 
(1) Potassium chloride 0.001M 0.0745 
(2) Di-Potassium  hydro gen Phosphate  (K2HPO4) 0.001 M 0.175 
(3) Calcium sulphate (CaSO4.2H2O) 0.002 M 0.344 
(4) Magnesium sulphate  (MgSO4.7H2O) 0.001 M 0.246 
(5) Trace  elements  solution:   0.5 ml 
1. Copper sulphate (CuSO4 5H2O) 0.01mg/kg 0.78 
2. Zinc Sulphate (ZnSO 4 7H2O) 0.25 mg/kg 2.22 
3. Ammonium  molyb date ((NH 4).6Mo7O24.4H2O) 0.0025 mg/kg 0.01 
4. Magnesium  sulphate (MgSO4.7H 2O) 0.25 mg/kg 2.03 
5. Boric  acid (H3BO4) 0.125 mg/kg  1.43 
6. Water  1000 ml 
Prep are the solution no (5) cons isting of trace elements  in one litre of stock solution and 
add to final nutrient solution at the rate of 0.5 ml per litre. 
(6) Iron solution   0.5 ml 
1. Ferrous sulphate   5 
2. Citric acid  5 
3. Water  100 ml  
Prep are the solution  no. (6) As 100  ml of stock solution and add final nutrient solution at the 
rate of 0.5 ml per litre. 
D.1.2  Preparation of plant nutrient solution  
D.1.2.1  Prepare the nutrient solution by weighing out substances (1), (2) and (4) and dissolving them  in a 
liter of water.  
D.1.2.2  To this solution add 0.5 ml of trace elements solution and 0.5 ml of iron solution. 
D.1.2.3  Grind in a mortar 0.344 g of calcium sulphate  (3) to a fine consistency  and add to the final nutrient 
solution. 
D.1.2.4  Autoclave the nutrient solution thus prepared, at 121 C for 15 min. 
NOTE 1  The nutrient solution may be prepar ed in the tap water provided  the water  is soft. 
NOTE 2  The nutrient solution should be shaken well to disperse  calcium sulphate before  dispensin g. DRS 488 -1: 2022 
13                ©RSB 2022  - All rights reserved  
 NOTE 3  If the solution  is made up with distilled water, the pH is about  7.2 before  autocl aving and falls to 5.5 on 
autoclaving and rises  slowly on standing to about 5.8. However, there  is no need to adjust pH. For most tropical legumes; pH 
of about  6.0 is adequate.  
D.2 Procedure for nodulation  
D.2.1  Immerse the seeds in 95 percent alcohol and follow by surface steri lization in freshly prepared 
chlorine water  (for 15 to 20 min) or 0.1 percent mercuric chloride solution 3 min in a suitable container 
such  as a screw -capped bottle or a test tube with a rubber hung.  
NOTE 1  In case of seeds with tough  seed  coat,  conc entrated  sulphuric acid may be used  as a surface  Sterilants  for 
20 to 30 min. 
NOTE 2  It is reco mmended that the seeds should be placed overnight in a desi ccators containing calcium chloride before 
surface  steril ization with sulphuric acid. 
D.2.2  Pour out the Sterilants  and wash the seeds in seve ral chang es of sterile water  and wash the see ds 
in seve ral chang es of sterile water (at le ast ten times) to get  rid of the Sterilants.  
D.2.3  Fill earthenware or glazed pot with soil (2 parts  soil and 1 part washed  coarse sand) (pH 6 to 7) and 
autoclave for 2 h at 120 C. 
D.2.4  After two-days  incubation at room temperature, repeat autoclaving to  ensure  comple te sterility of 
soil. Inoculate surface sterilized see ds with  water  slurry of the inoculan ts taken  from a culture packet (15 to 
100 g seeds per gram of inoculants depending on the size of the seed)  and sow the seeds.  Keep a set of 
pots with uninoculated seeds as contr ol and also a set of pots with ammoni um nitrate at the rate of 100 
kgN/ha as control aid incubate them in a pot‐culture house  during appropriate seaso ns for appropr iate plants, 
taking care to separate the inoculated  pots from the contr ol pots. 
NOTE  If growth rooms  or cabinets  having  facilities to adjust tempera ture and light are available, the pots may be 
incubated in such controll ed environmen tal conditions.  
D.2.5  Sterilize the nutrient s olution at 120 C for 20 min and irrigate each pot once to the moisture holding 
capacity of soil. Subsequently, water the seedling pe riodically with sterilized water preferably through a plastic 
tube, taking care to prevent splashing of water  from inoculated  pots to uninoculated  ones. 
D.2.6  Maintain required number of replicated p ots (4 to 6) for each bo tanical species for statistical analysis. 
D.2.7  After two to three weeks  of growth, thin down the number of plants in each pot to four uniform 
plants. 
D.2.8  At the end of 6 to 8 weeks,  take one set of pots from both the control and inoculated series and, 
sepa rate the plants carefully from the soil under  slow running water.  Obtain data on the number, colour 
(effective nodules are pink or red) and mass of nodules. 
D.2.9  At the end of 6 to 8 weeks, harvest the shoot system, dry at 60 C for 48 h and determine dry mass. 
For the above  purpose, maintain adequ ate replications of pots (4 to 16).  
©RSB 2022  - All rights reserved                  14 
 D.2.10  Record the nodulation data regarding for mation of  pink colour of  nodules as revealed visual ly when  
nodules are cut open  by razor blade. After computing  the data,  based  on the dry mass of plants and 
nodulation data decide the effectiveness of  culture. If  good effective pink nodulation is obtain ed in inoculated  
plants  together with local absence or sometimes presence of stray nodules in controls and if there is a 50% 
increase in the dry mass of plants over the uninoculated control  without nitrate, it may be concluded that 
the culture is of the required quality. DRS 488 -1: 2022 
15                ©RSB 2022  - All rights reserved  
 Annex  E 
(normative)  
 
Total plate count of Azotobacter  
E.1 Preparation of culture Medium  
E.1.1  Use a plating medium of the following composition 
Agar 20.0 g  
Sucrose (C12H22O11) 20.0 g  
Ferric sulphate Fe2 (SO4)3 0.1 g  
Dibas ic potassium  phosphate (K 2HPO 4) 1.0 g  
Magnes ium sulphate (MgSO4.7H 2O) 0.5 g  
Sodium  Chloride (NaCl) 0.5 g  
Calcium carbonate (CaCO 3) 2.0 g  
Sodium  Molybdate (Na2MoO4) 0.005 g  
Distilled water 1000 ml  
pH 6.8 to 7.2  
E.1.2  Use nutrient agar (Beef extract 3.0g, tryptone  5.0 g, agar 15.0 g and distilled water 1000 ml, pH 7.0) 
for total count of bacteria. The  extent of contamination can be assessed  by difference betw een the total 
bacterial  count  and Azotobacter chroococum coun t. 
E.1.3  The culture medium shall be prepared in accordance with ISO 11133 or using any other acceptab le 
validated test method.  
E.2 Preparation for serial  dilution  for plate  count  
E.2.1  Add 30 g of Inoculants to 270 ml of sterile  distilled or demineralized water  and shake  for 10 min on 
a recipro cal shaker or homogen izer. 
E.2.2  Make serial dilutions up to 10-9. Take 0.1 ml or suitable aliquots of 10-6 to 10-9 dilutions using sterile  
pipettes and deliver to Petri dishes contai ning set medium as given in E.1.2  and spread it uniformly with a 
spreader. Invert the plate s and promptly place them  in the incubator. 
E.3 Incubation of plates  
Label the plates  and incubat e at 28±1  C for 4 to 6 days. 
E.4 Colony counting aids  
Same as in C.4. (annex C)   
©RSB 2022  - All rights reserved                  16 
 E.5 Colony count  
E.5.1  Azotobacter chroococcum colonies are gummy, raised with  or with out striations, visco us 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 num ber per  gram of the carrier material. 
E.5.2  Grow the vegetative c ells at 30 C on Burks  agar medium comprising sucrose 20 g, dipotassium 
hydrogen p hosphate 0.64 g, dihydr ogen potassium ph osphate 0.20 g, sodium chloride  0.20 g, calcium 
sulphate 0.05 g, so dium molybdate 0.0 01 g, ferric sulphate 0.003 g, agar 20 g and distilled wa ter 1000 ml. Look 
for vegetative c ells after 18 to 24 h either by simple staining method or through  a phase contrast  microscope. 
E.5.3  Grow the cyst cells on Burks  agar medium as given above with 0.3% n-butanol in place of the 
carbon source. L ook for cyst formation after 4 to 5 days incubation. DRS 488 -1: 2022 
17                ©RSB 2022  - All rights reserved  
 Annex  F 
(normative)  
 
Test for nitrogen fixation in pure culture  
F.1 Preparation of pure culture medium  
Prepare medium as given in E.1, excluding agar. 
F.2 Procedure  
Select from each Azotobacter colony, of the type that has been counted as Azotobacter chroococcum. Pick 
up one colony  and plate on the medium given  in (E.1). Use this pure culture  for inoculating the broth  for 
nitrogen  fixation. For this purpose, take 50 ml aliquots of broth in 250 ml conical flasks for inoculation. 
Inoculate and incubate for 7 days on a rotary shaker at 29 ± 1 C.  Test the contents  of the flasks for purity 
by streaking on fresh medium. Concen trate the contents  over water bath (50 to 60 C) or in an oven  at 90 
C or under vacuum  at 60 C to dryness. 
Collect the dried culture and take it as a sample. 
The contents of the flasks in inoculated control series (flasks inoculated and kept in deep freeze to inhibit 
growth) should be prepared in a similar manner to serve  as control. 
F.3 Determination by Kjeldahl method  
F.3.1  Reagents  
F.3.1.1  Sulphulic acid, 93 – 98 %, N -free 
F.3.1.2  Mercuric oxide, N -free 
F.3.1.3  Sodium hydroxide solution, N -free: Dissolve about 450 g of solid sodium hydroxide in water, cool and 
dilute to 1 L (specific gravity of the solution s hould be at least 1.36)  
F.3.1.4  Potassium  sulphate  (or anhydrous  sodium sulphat e) 
F.3.1.5  Sulphide  or thiosulphate solution: Dissolve 40g commer cial potassium sulphide in 1 L of water (or 
solution of 40g of sodium sulphide or 80g of sodium thiosulpha te in 1L may be used). 
F.3.1.6  Zinc granu les‐reagent grade.  
F.3.1.7  Indicators  
©RSB 2022  - All rights reserved                  18 
 F.3.1.8  Methyl  red indicator: Dissolve 1g of methyl red in 200 ml of Ethanol. 
F.3.1.9  Mixed indicator: Prepare  mixed indicator by Dissolving 0.8 of methyl red and 0.2 g of methyl blue 
in 500 ml of ethanol. 
F.3.1.10  Hydrochlo ric or sulphuric acid: Standa rd solution 0.5 N or 0.1 N when amount of nitrogen  is small. 
F.3.1.11  Boric  acid, 4% solution 
NOT E Ratio of salt to acid (m/v) should be abou t 1:1 at the end of the digestion  for proper  temperatu re control.  
Digestion may be incomplete  at a lower ra tio, and nitrogen may be lost at higher  ratio. Each gram of  fat consu mes 10 ml 
of sulph uric acid and each  gram  of carbohydrate  4.0 ml of sulphuric acid during digestio n. 
F.3.2  Apparatus  
F.3.2.1  For digestion  
F.3.2.1.1   Use kjeldahl’s  flasks of hard, moderat ely thick, well annealed glass with total capa city 
appro ximately 500 to 800 ml. Conduct diges tion over heating  device adjust to bring 250 ml of water at 25 
C to rolling boil in about  5 minutes. To test the heaters, preheat for 10 minutes in the case  of gas burners 
and for 30 minutes in the case of electric heaters.  
F.3.2.1.1   Add 3 to 4 boiling chips to prevent superheating. 
F.3.2.2  For distillation  
F.3.2.2.1  Use 500 to 800ml Kjeldahl’s flask fitted with rubber  stopper through  which  passes the lower  
end of an efficient scrubber bulb or trap to prevent mechanical carry‐over of so dium hydroxide during 
distillation. 
F.3.2.2.2  Connect  the upper end of the bulb tube to a condenser by rubber  tubing. 
F.3.2.2.3  Trap the out let of the condenser  in such a way as to ensure  absorption of ammonia distilled 
over with the receiver. 
F.3.3  Procedure  
F.3.3.1  Place 0.25 g  of the sampl e in the d igestion flask. Add 0.7 gm mercuric oxide, 15 gm p otassiu m 
sulphat e followed by 25 ml of sulphuri c acid. Shake, let stand for abou t 30 minute s and heat carefull y until 
frothing ceas es. Boil briskl y until the solution clears  and continue  boiling further for 90 minutes.  
F.3.3.2  Cool,  add about 200 ml of water cool to room  tempera ture and add a few zinc granules.  
F.3.3.3  Tilt the flask and carefully add 50 ml of sodium hydroxide solution without agitation.  Immediate ly 
connect the flask to the  distillation bulb on the condenser whose tip is imme rsed in 50 ml of standard 0.1 N 
acid in the receiving flasks. Ro tate the  digestion flask carefully to mix the conten t. Heat  until 150 ml of the 
distillate collects and titrate excess acid with 0.1 N base using methyl‐red or mixed indicator. Carry  out blank 
determinati on on reagen ts. DRS 488 -1: 2022 
19                ©RSB 2022  - All rights reserved  
 NOTE Check  the ammonia  recording periodically, using inorganic  nitrogen control, for example, ammoni um sulphate. 
F.3.4  Calculation  
Nitrogen content,  (% by mass) = 
m1.4N T.V.  
Where;  
  T.V.  titration value (ml of standard acid)  
  N  normality of standard acid, and  
  M  mass of sample taken.  
F.4 Determination of sucrose consumed  
F.4.1  Sample preparation  
F.4.1.1  Take 1g of accurate ly weighed prepared sample of AI (F.2)  into 250 ml volumetric flask and dilute 
with about  150 ml of water. 
F.4.1.2  Mix thoroughly the contents  of flask and make the volume to 250 ml with water.  Centrifuge  the 
mixture  for 15 min at 10 000 rpm. 
F.4.1.3  Decant  the supernatant carefully and estimate the total reducing sugar after inversion (F.4.2) 
F.4.2  Method of inversion  
F.4.2.1  To 100 ml of the super natant (F.4.1) add 1 ml of concen trated  hydrochlo ric acid and heat the 
solution to near boiling. Keep aside overnight.  
F.4.2.2  Neutralize  this solution with sodium carbonate and determined  the total reducing sugars. 
F.4.3  Determination of reducing sugars  
F.4.3.1  Reagents  
F.4.3.1.1  Copper sulphat e solution  (Solution A): Dissolve 34.639 g of copper  sulphate crystals 
(CuS O4.5H5O) in water , dilute to 500 ml and filter through  glass wool or filter paper.  
F.4.3.1.2  Potassium  sodium tatrate (Rochelle  salt) (Solution B): Dissolve 173g of potassium sodium 
tartrate and 50g of sodium hydro xide in water,  and diltute to 500 ml.  Let the solution  stand for a day and 
filter.   
©RSB 2022  - All rights reserved                  20 
 F.4.3.1.3  Hydroch loric acid, specific gravity 1.18 at 20 C (appr oximately 12N)  
F.4.3.1.4  Standard invert sugar solution 
F.4.3.1.4.1  Weigh  accurately 0.95g  of sucrose and dissolve it in 500 ml of water.  
F.4.3.1.4. 2 Add 32 ml of concen trated  hydrochloric acid, boil gently  for 30 min and keep  aside for 24 h. 
F.4.3.1.4. 3 Neutralize  with sodium carbonate  and make  the final volume  to 1000 ml, 50 ml of this solution 
contains 0.05g of invert  sugar. 
F.4.3.1.5  Methylene blue indicator ‐ 0.2% in water  
F.4.3. 1.6 Standardization of copper  sulphate solution: 
F.4.3.1.6.1  Using separate pipettes, pipette accurately 5 ml of solution A  (F.4.3.1  a) and 5 ml of solution  
B (F.4.3.1  b) into a conical flask of 250 ml capacity. 
F.4.3.1.6.2  Heat  this mixture to boiling on asbestos gauze and add standard invert sugar solution (F.4.3.1 
d) from a burette, about  1 ml less than the expected volume, which  will reduce the Fehling solution 
complet ely (about  48 ml). 
F.4.3.1.6.3  Add 1 ml of methylene blue indicator while keeping the soluti on boiling. Complete the titration  
within 3 min. The  end point being indicated by change  of colour from blue to red.  
F.4.3.1.6.4  From  the volume  of invert sugar  solution used,  calculate the streng th(S) of the copper sulphate 
solution  by multiplying the titre value  by 0.001 (g/ml of the standard invert sugar solution). This would give 
the quantity of  invert sugar requ ired to reduce the copper  in 5 ml of copper  sulphate solution. 
F.4.3.2  Procedure  
F.4.3.2.1 Using se parate pipe ttes, pipette ac curately 5 ml of s olution A  (F.4.3.1 a) and 5 ml of solution B 
(F.4.3.1 b) in porcelain dish. 
F.4.3.2.2 Add abour 12 ml of AI solution from a burette  and heat to boiling over a gauz e. 
F.4.3.2.3 Add 1 ml of methylene  blue indicator  and while  keeping the solution  boiling comp lete the 
titration  within 3 minutes, the end point being by change  of colour from blue to red. 
F.4.3.2.4 Record the volume (H) in ml of AI solution required for titration.  
F.4.3.2.5 Follow  the same step steps for control. DRS 488 -1: 2022 
21                ©RSB 2022  - All rights reserved  
 F.5 Calculation  
F.5.1  Total reducing sugars  
Total reducing sugars, (% m/m) = 
MHS 100 250
  
Where,  
  S strength of copper sulphate solution  
  H volume in ml of Al solution required for titration, and  
  M mass in g of Al taken for the test  
F.5.2  Determination of sucrose  
.95 (%m/m)] inversion) before sugars (reducing - (%m/m) inversion) after sugars [(reducing  m/m) (% Sucrose  =
 
F.5.3  Determination of nitrogen fixed per g of sucrose consumed  
Nitrogen fixed (mg)/g sucrose consumed = 
(F.5.2) consumed Sucrose(F.3.4) (%m/m) content Nitrogen   
©RSB 2022  - All rights reserved                  22 
 Annex  G 
(normative)  
 
Determination of the number of Azospirillum cells  
G.1 Medium  
Use N‐free semisolid medium (Nfb) of the following composition for preparation of MPN  tubes 
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  
Bromothymol blue (0.5 % alcoholic solution)  2.0 ml  
Agar  1.7 g  
pH Adjusted to 6.5 – 7.0 
G.2 Preparation of serial dilution for MPN count  
G.2.1  Dispense 30  g of Azosp irillum inoculants in 270 ml of sterile  water  and shake  for 10 minu tes on a 
recipr ocal shaker. Make seria l dilution s up to 10-7 dilution.  
G.2.2  Pipett e out 0.2 ml aliquots of 10-4 to 10-8 dilution and delive r it to screw  cap tubes  or petridishes 
contain ing set medi um as described in E‐2.1. Spre ad the aliquots over the plate s. Inve rt the plates and place 
them in the incubator at 28 ± 2°C for 3 days .  Use 5 replicates of 10-5, 10-6 and 10-7 level.  
G.3 Counting  
G.3.1  Count the tub es or plate s which have turned blue in colour after inoculation and ascertain the presence 
of pellicle s in undisturbed medi um. To determine usual contam ination on the same exami ne doubtfu l objects 
carefully.  
G.3.2  Count  all plates/ tubes which have turned  blue and consider them  for the purpos e of calculation . Count 
such type of tubes/plates  and tally this count with MPN table to get the numbe r of cells per gram  of the carrier.  
Azos pirillum count/g  of carrier = 
product of massDry level Dilution  table MPN from Value   
 DRS 488 -1: 2022 
23                ©RSB 2022  - All rights reserved  
 Annex  H 
(normative)  
 
Test for effective root growth  
H.1 Pot Culture Test  
H.1.1  For the purpose of 'Pot Culture Test' plant nutrition solution, preparation of solutions and procedure 
shall be same as described in C.2.1.1 and C.2.1.2 of Annex C.  
H.1.2  After three weeks of growth, thin down the number of plants in each pot to four uniform plants. At the 
end of three months, take one set of pot from control and inoculated series and separate the plants carefully 
from the soil under slow running water. Obtain the number, length and mass of the plant as a whole including 
branch, s eeds if any and roots.  
H.1.3  At the end of three months, harvest the shoot system (depending on the crop). Dry at 60 °C for 48 hours 
and determine the dry mass. For the above purpose maintain a minimum of four replications or a maximum of 
16 replications.  
H.1.4  Better vigour of roots in inoculated seeds if established from the data when compared to the control, is 
the confirmation of effectiveness of ASI. If there is 10% increase in the plants over the dry mass of uninoculated 
control without nitrate, it ma y be concluded that the culture is of required quality.  
H.2 Method for estimating MPN count for Azospirillum  
H.2.1  Add 100 g ASI sample to 900 ml of sterile distilled water.  
H.2.2  Shake  for 10 minutes  on a reciproca l shaker. 
H.2.3  Make  ten-fold dilution series. 
H.2.4  Pipette 1  ml of each dilution (from 10-1 to 10-7) to each one of  5 replicates (5  tubes containing 
nitrogen  free bromothymol  blue semi‐solid malate media for Azospir illum). 
H.2.5  Begin by taking  aliquot from the highest dilution and proceed down  the series with same pipette 
H.2.6  Collect the tubes  from 10-5, 10-6 and 10-7 dilution level. 
H.2.7  Incubate  the inoculated  series for 2 days at 30°C. 
H.2.8  Check  the characteristic sub‐surfac e while particles (+ or ‐) in malat e semi‐solid medium, a change  to 
dark blue colour of the medium.   
©RSB 2022  - All rights reserved                  24 
 H.3 Calculation  
H.3.1  To calculate  the most  probable numbe r of organism s in the original sample , select as P1 the nu mber of 
positiv e tubes  in the least concentrated dilution  in which all tubes  are positive or in which  the greates t numbe r 
of tubes  is +ve, and let P2 an d P3 represent  the numbe rs of positive tubes  in the next two higher dilution.  
H.3.2  Then find the row of number s in Table E.1 in which P1 an d P2 correspond to the values observ ed 
experimenta lly. Fol low that row of number s across the table  to the column heade d by the observed  value of P. 
H.3.3  The figure at the point of intersection  is the most  probable numbe r of organisms in the quantit y of or iginal 
sampl e repr esented in the inoculum adde d in the  second dilution. Multi ply this figure by the appropriate dilution 
facto r to obtain the MPN  value.  
Tables H.1 — Most  Probab le Num bers for use with 10 Fold  dilutions  and S Tube s per dilution 
(Cochran, 1950) 
 
DRS 488 -1: 2022 
25                ©RSB 2022  - All rights reserved  
 Annex  I 
(normative)  
 
Guidelines on maintenance and preparation of culture and quality control at broth 
stage  
I.1 Rhizobium  
I.1.1 Maintenance of pure cultures  
I.1.1.1  Maintain pure culture of rhizobia on yeast extract  mannitol agar (YEMA) slants to the following 
composition. 
Mannitol  10.0g  
Potassium  hydrogen phos phate (K2HPO 4) 0.5 g  
Magne sium sulphate (MgS O4 7H2O) 0.2g  
Sodium chloride  (NaC l) 0.1 g  
Calci um carbonate (CaCO3) 1.0 g  
Yeast extracts 1.0g  
Agar 18g  
Distilled water  1000 ml  
pH 6.8 ‐ 7.0 
I.1.1.2  Transfer  a loopful of the pure culture  to each  of the agar slant  asep tically in an inoculation room 
and incubate at 28 ± 2 C for 3 to 10 days depending upon the species of Rhizobium. Always keep  culture 
at 4 C. 
I.1.2 Preparation of inoculum cultures  
I.1.2.1  Prepare yeast extract mannitol broth of the composition as given in J.1.1 minus the agar.  
I.1.2.2  Weigh  the ingred ients, pool, dissolve in required amount of water and make upto known  volume.  
Dispense 100 ml aliquots of the broth into 250 ml conical flasks and plug with non‐absorbent cotton. 
Autoclave the flasks at 121 C for 15 min, cool and inoculate  with the required Rhizobium as given in J.1.2 
(c). 
I.1.2.3  Examine a 4 to 6 days  old pure culture visually and by Gram  staining te st for freedom from 
contamination and transfer approximately 2  to 3 ml of sterile broth aseptically into it. Scrape the bacterial 
growth with the help of an inoculation needle and trans fer the resulting bacterial susp ension into  a 250 ml 
conical flask containing the  broth. Incubate the flasks at 28 ± 2 C on a rotary shaker for 2 to 6 days 
depending upon  the species of the Rhizobium. 
I.1.3 Preparation of mass culture  
Form a battery of conical flasks or suitable ferment ors each  conta ining requir ed amounts  of broth (see J.1.2  
(a)) and proceed  for sterilization (see J.1.2 (b)). Into each  one of these  flasks, transfer requisite quantity of  
©RSB 2022  - All rights reserved                  26 
 inoculum culture  aseptically and incubate the flasks preferably on  a rotary shaker at 28 ± 2 C for 2 to 6 
days depending on the species of the Rhizobium. 
NOTE  If the medium (broth) is formulated in a fermentor  the amount  or inoculum culture to be transferred into the 
fermentor  is variable depend ing on the size of the ferment or but the ratio between  the inocul um culture and the medium 
formulated  in the fermentor  is recommended as I:20 (inoculum :quality of broth  ) in the fermento r. 
I.1.4 Quality control tests recommended at broth stage  
I.1.4.1  Qualitative tests  
I.1.4.1.1  Check for freedom from the visible contaminants 
I.1.4.1.2 The pH of the bacte rial broth shall normal ly be between  6.5 and 7.5.  
I.1.4.1.3 Smear  and Gram  stain  
a) Reagents  
1) 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 amm onium oxalate in 30 ml of distilled  water.  
Mix the two solution and filter through a filter paper. 
2) Iodine solution 
Iodine  1.00 g  
Potassium iodide  2.00 g  
Distilled water  300 ml  
Weigh the ingredient  and dissolve in water. Filter through a filter paper. 
3) Erythrosine  
Erythrosine  1.00 g  
Phenol 5.00 g  
Distilled  water 100 ml  
Weigh the ingre dient, dissolve in distilled water and filter through a filter paper. 
b) Procedure  
 DRS 488 -1: 2022 
27                ©RSB 2022  - All rights reserved  
 Prepare a smear on a clean microscope  slide, fix over a flame by gently and intermit tent heating, air cool 
and flood  with ammonium, oxalate crystals violet stain for 1 min. After removing the excess  of ammonium 
oxalate  crystals violet, wash the slide under a gen tle stream of running tap water. Flood the slide with iodine 
solution for half a minute remove excess stain wash  95% ethyl alcohol and finally wash under a gen tle stream 
of running tap wa ter. Flood the slide with erythrosine stain for about 3 min, wash under a gentle stream of  
running tap water and dry betw een the folds of a filter paper. Examine  the slide under a compound microscope 
using an oil imme rsion objective. 
NOTE  A smear prepared from undiluted broths should be free from Gram  positive cells. The presence  of a few gram  
positive cells in occa sional fields  which may be due to dead cells in the medium may be disregard ed. 
I.1.4.1.4  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 (NaCl) 5.0 g  
Agar  15.0 g  
Distilled water 100 ml 
Bromocresol  purple 10 ml of ethyl alcohol solution (1.6%) 
pH 7.2 
NOTE  When a loopful  of the broth  is streaked in to this Medium and incubated at  28 ± 2 C for 24 h, the p urple‐violet 
colour  of the mediu m (due to t he ind icator bromo‐cresol  purple) shall not change.  If the colour changes  to yellow (acidic 
reacti on) or blue (alkaline reaction) the broth is gross ly contam inated.  Hence the broth should  be rejected. 
I.1.4.1.5  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± 2 C for 3 ‐ 10 
days, it shows colonies of bacteria  with growth characteri stics same as that of the pure culture used in the 
preparation of the broth, otherwise  the broth  should be rejected.  
J.1.4.2 Quantitati ve test 
Viable  or plate counts  
Serial ly dilute one milliliter of the broth to obtain dilutions of the order of 10-6 to 10-9. Plate 0.2 ml aliquots 
of the dilution 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 fermenters  shall be not 
less than 10-8 to 10-9 cells/ ml. Other wise, the broth should be rejec ted. 
I.2 Azospirillum  
I.2.1 Maintenance of pure cultures  
I.2.1.1  Maintain pure culture of Azospirillum on nitrogen free  bromothymol  blue medium and maintain 
as solid medium.  
©RSB 2022  - All rights reserved                  28 
 I.2.1.2  Transfer a l oopful 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. 
I.2.2 Preparation of inoculums culture and mass culture  
Inoculums culture and mass culture  of this standard shall be prepared as described for H.1. 
I.2.3 Quality control test recommended at broth stage  
I.2.3.1  Quality test  
I.2.3.1.1  Check for free from contaminants by preparing slide and observing under microscope.  
I.2.3.1.2  The pH of bacterial  broth shall normally be betw een 7.0 to 8.0. 
I.2.3.1.3  Gram  staining test shall be carried out as described in J.1.4.1.3. 
I.2.3.1.4  See the col our change in the media after 24  hours from i noculation. The colour will change from 
green  to blue. 
I.2.3.1.5  Watch the pellicle just below the surface of the media. It is checked on the third day after keeping  
inoculated  broth undisturbed.  
I.2.3.2  Quantitative test  
Most Probable Number (MPN)  
I.3 Azotobactor  
I.3.1 Maintenance  
I.3.1.1  Maintain pure culture of Azotobacter on slants of the following composition 
Agar  20 g  
Sucrose 20 g  
Ferrous Sulphate 0.1 g  
Dibasic  Potassium  Phosphate 1.0 g  
Magn esium Sulphate 0.5 g  
Calcium carbonates 2.0 g Sodium   
Molybdate  0.005 g 
I.3.1.2  Transfer a loopful pure culture to each of  agar slants aseptically in an ino culation room and incubate 
at 28 ± 2 C for 3 to 10 days depending up on the species of Azotobactor. Always  keep pure culture at 5 
C. DRS 488 -1: 2022 
29                ©RSB 2022  - All rights reserved  
 I.3.2 Preparation of inoculums culture  
I.3.2.1  Prepare Jensen's  media broth of the compositor  as given in J.3.1 minus the agar.  
I.3.2.2  Transfer a loop full of the culture into a 100 ml/250  ml conical flask containing the broth. Incubate  
the flasks at 28 ± 2 C on a rotary shaker for 2 to 6 days. 
I.3.3 Quality control tests recommended at broth stage  
I.3.3.1  Quality test  
I.3.3.1.1  Check for free from contaminants by preparing slide and observing. 
I.3.3.1.2  The pH by bacterial broth shall normal ly be between 7.0 to 8.0.  
I.3.3.1.3  Gram  staining test shall be carried out as described in J.1.4.1.3. 
I.3.3.1.4  See the col our change in the media after 24  hours from i noculation. The  colour  will change from 
green  to blue. 
I.3.3.1.5  Watch the pellicle just below the surface of the media. It is checked on  the third day after keeping  
inoculated  broth undisturbed.  
I.3.3.2  Quantitative test  
Most  Probable Number  (MPN)  
©RSB 2022  - All rights reserved                  30 
 Bibliography  
[1] IS 8268:2001, Rhizobium inoculants  — Speci fication 
[2] IS 9138:2009, Azotobac ter chroococcum  inoculants — Specification 
[3] IS 14806: 2000, Azospirllum inoculants — Specification 
[4] Biofert ilizers  and Orga nic Fertilizers in Fertilizer (Contro l) order, 1985, published by National 
Centre of Organic  Farming,  Department of Agriculture and Coop eration, Ministry of Agriculture, Govt  of India. 
 
 DRS 488 -1: 2022 
 
Price based on  nnn pages  
©RSB 2022 - All right s reserved