DEAS 1176 :2023  
ICS 65.080  
© EA C 2023                                                                                                                          First Edition 2023   
 
DRAFT EAST AFRICAN STANDARD  
      Biofertilizers  — Specifications  
EAST AFRICAN COMMUNITY  
 
 
 
 
 
 
 
 
   
 DEAS 1176:2023  
ii © EAC 2023  – All rights reserved  
 Copyright notice  
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Reproduction for sales purposes may be subject to royalty payments or a licensing agreement. 
Violators may be persecuted  DEAS 1176:2 023 
© EAC 2023  – All rights reserved  iii 
 Foreword  
Development of the East African Stan dards has been necessitated by the need for harmonizing requirements 
governing quality of products and services in the East African Community. It is envisaged that through 
harmonized standardization, trade barriers that are encountered when goods and servi ces are exchanged 
within the Community will be removed.  
In order to achieve this objective, the Community established an East African Standards Committee mandated 
to develop and issue East African Standards.  
The Committee is composed of representatives o f the National Standards Bodies in Partner States, together 
with the representatives from the private sectors and consumer organizations. Draft East African Standards 
are circulated to stakeholders through the National Standards Bodies in the Partner State s. The comments 
received are discussed and incorporated before finalization of standards, in accordance with the procedures 
of the Community.  
East African Standards are subject to review, to keep pace with technological advances. Users of the East 
African Standards are therefore expected to ensure that they always have the latest versions of the standards 
they are implementing.  
The committee responsible for this document is Technical Committee EASC/TC 020, Agriculture and 
agrochemicals.  
 DEAS 1176:2023  
 
  Introduction  
Biofertilizers are substances, which contain living microorganisms, which, when applied to seed, plant 
surfaces, or soil, colonize the rhizosphere or the interior of the plant and promote growth by increasing the 
supply or availability of nutrients to the host  plant.  
 
Biofertilizers add nutrients through the natural processes of fixing atmospheric nitrogen, solubilizing 
phosphorus or nutrient mobilization to stimulate plant growth through the synthesis of growth promoting 
substances. They can be grouped in diff erent ways based on their nature and function.  
 
Most biofertilizers are produced from microorganisms such as Rhizobium, Azotobacter, Azospirillum, 
phosphate -solubilising bacteria. Other types include Mycorrhizal biofertilizers, potassium -mobilizing 
biofert ilizers, lactic acid producing bacteria and zinc -solubilising biofertilizers. The use of biofertilizers 
offers economic and ecological benefits by way of soil health improvement and fertility.  
 
Biofertilizers, known as microbial products, act as nutrient s uppliers and soil conditioners that lower 
agricultural burden and conserve the environment. Good soil condition is imperative to increased crop 
production, as well as human and/or animal health welfare. Thus, the materials used to sustain good soil 
conditi on, are treated as environmental matters. However, there are still some problems to be met on the 
use of microbial products. More precise quality control should be made in favour of the customers. With 
this in mind, we will do our best to develop better pr oduction techniques and to improve the management 
system for microbial products.  
 
Although the effects of biofertilizers are different among countries  due to variances in climate and soil 
conditions, the importance of biofertilizers on environmental conser vation in the 21st century should not be 
ignored. In the same manner, variou s acceptable  biotechnologies can be used  for increasing the 
biofertilizers effects with concern for the environment.  
 
The objective of this standard is to ensure that biofertilize rs on the market are appropriately tested 
through the quality criteria provided while ensuring that farmers obtain only certified products and as well 
aid the industry in the manufacture of quality biofertilizers. This standard will also promote the safe u se of 
biofertilizers and promote fair trade  
 
 
 
 
 
 DEAS 1176:2023  
6 © EAC 2023  – All rights reserved  
 Biofertilizer — Specification  
1 Scope  
This draft East African  standard specifies requirements, methods of sampling and test for biofertilizers. 
This draft standard covers  the following types of biofertilize rs; Rhizobia, Phosphate solubilizing 
microorganism, Azospirillum and Azotobacter.  
 
2 Normative references  
 
The following referenced documents are indispensable for the application of this standard. For dated 
references, only the edition cited applies. For  undated references, the latest edition of the referenced 
document (including any amendments) applies.  
 
ISO 8157 , Fertilizers , Soil Conditioners  and beneficial substances  – Vocabulary  
 
AOAC 2006.03, Arsenic, cadmium, cobalt, chromium, mead, molybdenum, ni ckel, and selenium in 
fertilizers — Microwave digestion and inductively coupled plasma -optical emission spectrometry  
 
AOAC 965.08, Water (free) in fertilizers — Vacuum -desiccation  
 
ISO 8397, Solid fertilizers and soil conditioners – Test sieving  
 
ISO 483 2, Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of 
coliforms — Colony -count technique  
 
ISO 10390, Soil, treated biowaste and sludge — Determination of  pH 
 
ISO 11465, Soil quality — Determination of dry matter and w ater content on a mass basis — Gravimetric 
method  
 
ISO 5315, Fertilizers — Determination of total nitrogen content — Titrimetric method after distillation  
 
ISO 15958, Fertilizers — Extraction of water soluble phosphorus  
 
ISO 14820 -1, Fertilizers and liming  materials — Sampling and sample preparation — Part 1: Sampling  
 
ISO 14820 -2, Fertilizers and liming materials — Sampling and sample preparation — Part 2: Sam ple 
preparation  
 
 
 
3 Terms and definitions  
For the purposes of this standard, the terms and defin itions given in ISO 8157  and the following shall 
apply.  
 DEAS 1176:2023  
 
  3.1 biofertilizer  
any substance which contains live microorganisms, part of microorganisms or product of microorganisms 
in their natural or modified  forms that is manufactured, sold or represented f or use in the improvement of 
the condition of soils or to aid plant growth or crop yield  
3.2 efficacy  
 the ability of a fertilizer or supplement to fulfil any label claims and to produce a desired or intended result 
based on the labelled guarantees and di rections for use  
3.3 efficacy assessment  
evaluate product performance in order to establish appropriate label claims, active ingredient guarantee 
and usage patterns  
3.4 active agent  
living agent in a biofertilizer to which the improvement of the condit ion of soils , nutrient availability  or aid in 
plant growth or crop yield are attributed  
 
3.5 inoculants  
 
 preparations containing beneficial micro -organisms in a viable state, intended for increasing the number 
of desired microorganisms.  
 
3.6 bio fertilize r carriers  
 
 substances which support , present  and preserve  microorganisms in a viable state  
 
3.7 biological nitrogen fixation  
 
a process by which the atmospheric nitrogen gas is converted into forms that are easily absorbed by 
plants through biological pr ocesses.  
 
3.8 nodules  
 
swellings or outgrowths on roots and stems particularly of leguminous plants resulting from the activity of 
some atmospheric nitrogen fixing microorganisms.  
 
3.9 plant growth promoters/enhancers/phytohormones  
 
 substances which stim ulate the growth and development of plants  
 
3.10 symbiotic microorganisms  
 
 microorganisms that form a mutual relationship with plants.  
 
3.11 mineralization  
 
 biological breakdown of organic materials to release inorganic nutrients for plant uptake  
 
 
 
 DEAS 1176:2023  
8 © EAC 2023  – All rights reserved  
 3.12 immobilization  
 
process whereby inorganic nutrients are locked up in microbial cells thus rendering them unavailable to 
plants  
 
3.13 solubilization  
 
a process, where bound nutrients are dissolved by microbial action.  
 
3.14 substrate  
 
any material that se rves as source of nutrient for microorganisms.  
3.15 Strain  
specific variant or subpopulation of a microorganism that exhibits distinctive characteristics, often as 
result of genetic or environmental differences  
 
 
4 Requirements  
4.1 General requirements  
4.1.1 Biofertilizer shall contain effective strain in minimum recommended population.  
4.1.2 Biofertilizer shall not contain more than the maximum allowed contaminating microorganisms.  
4.1.3 Biofertilizer shall not contain genetically modified microorganisms  
4.1.4 Biofertilizer shall have single or a combination of effective strains.  
4.1.5 Biofertilizers shall contain no pathogenic organisms which could affect plants, animals and human  
beings.  
4.1.6  Biofertilizers carriers shall be of a nature that is not har mful to the environment  
4.1.7 Biofertilizer shall have at least one of the following properties:  
a) ability to fix nitrogen;  
b) ability to solubilize minerals;  
c) ability to improve plant performance.;  
4.1.8  
 
The use of biofertilizers shall take into cons ideration  the effect  of the applied  inoculants against inherent 
beneficial microbiome . DEAS 1176:2023  
 
  4.2 Specific requirements  
4.2.1  
Bio fertilizer shall comply with the following requirements based on the declared active agent as stipulated in Table 1 - 
Table 4.  
 
Table 1 — Specific requirements of rhizobia  containing biofertilizer  DEAS 1176:2023  
10 © EAC 2023  – All rights reserved  
 SL/No.                 Parameter                              Requirement  Test Methods  
i. Base  Carrier; solid or liquid based   
ii. Viable cell count 
(CFU)  
15 days after 
manufacturing and 
15 days before 
expiry date  Minimum 5x107 cell/g of carrier 
material or 108 cell/mL of liquid   Annex E 
iii. Contaminating 
microorganisms  
level No contamination at 105 dilution  Annex E  
iv. pH 6.5 - 7.5 ISO 10390  
v. Particle size(solid 
carrier -based)  All m aterial shall pass through 
0.15-0.212 mm IS sieve  ISO 8397  
vi. Moisture, % by 
weight, max.  (solid 
carrier -based)  30% - 40% AOAC 965.08  
vii. Nodulation test  Should be positive  Annex F  
viii. Efficiency character  Should show effective 
nodulation on all sp ecies 
(crops) listed on the packet 
nodulation (should appear pink 
when dissected)  Annex F  
    
ix. Shelf life  Should have at least 6 months  
for solid carrier -based 
inoculants and 6 weeks for 
liquid -based inoculants (under 
refrigeration) from date of 
manu facturing   DRAFT EAST AFRICAN STANDARD  DEAS 1176:2023  
 
© EAC 2023  – All rights reserved  1 
  
Table 2 — Specific requirements of phosphate solubilizing microorganisms  
containing biofertilizers  
SL/No.                 Parameter                              Requirement  Test Methods  
i. Base  Carrier; solid or liquid based   
ii. Viable cell  count (CFU)  
15 days after manufacturing and 
15 days before expiry date  Minimum 5x107 cell/g of carrier material or  
108cell/ mL of liquid   Annex G 
iii. Contaminating microorganisms 
level No contamination at 105 serial dilution  Annex G  
iv. pH 6.5 - 7.5 (for solid) 5 - 7.5 (for liquid)  ISO 10390  
v. Particle size  (solid carrier -based)  All material shall pass through 0.15 -0.212 
mm IS sieve  ISO 8397  
vi. Moisture, % by weight, max. ( solid 
carrier -based)  35% - 40 %  AOAC 965.08  
vii. P –solubilization  30% - 50 % Annex H  
viii. P- solubilisation zone , min  1 mm  Annex G  
ix. Shelf life  Should have at least 6 months  for solid 
carrier -based inoculants and 6 weeks for 
liquid -based inoculants (under refrigeration) 
from date of manufacturing   
 
Table 3 — Specific requi rements of azospirillum  containing biofertilizer  
SL/No.                 Parameter                              Requirement  Test Methods  
i. Base  Carrier; solid or liquid based   
ii. Viable cell count (CFU)  
15 days after 
manufacturing and 15 
days before expi ry date  Minimum 5x107 cell/g of solid carrier material 
or108cell/mL of liquid  Annex I 
iii. Contaminating 
microorganisms  level No contamination at 105 serial dilution  Annex I   
iv. pH 7.0 – 8.0  ISO 10390  
v. Particle size(carrier -
based)  All material shall  pass through 0.15 -0.212 
mm IS sieve  ISO 8397  
vi. Moisture, % by weight, 
max. (carrier -based)  35% - 40 %  AOAC 965.08  
vii. Efficiency Character   Formation of white pellicle in semi solid 
nitrogen free bromothymol blue media.  
1.  
Shall show effective root dev elopment on all 
cultivar/crops  Annex J  
viii. Shelf life  Should have at least 6 months for  solid 
carrier -based inoculants and 6 weeks for 
liquid -based inoculants (under refrigeration) 
from date of manufacturing   
ix. P- solubilisation zone  1 mm  Annex G  
 DEAS 1176:2023  
2 © EAC 2023  – All rights reserved  
 Table 4 — Specific requirements of Azotobacter  containing biofertilizer  
SL/No.                 Parameter                              Requirement  Test methods  
i. Base  Carrier; solid or liquid based   
ii. Viable cell count  (CFU)  
15 days after 
manufacturing a nd 15 
days before expiry date   Minimum 5x107 cell/g of solid carrier 
material or108cell/mL of liquid   Annex  K 
iii. Contaminating 
microorganisms level  No contamination at 105 dilution.  Annex K  
iv. pH 6.5 – 7.5 ISO 10390  
v. Particle size(carrier -
based)  All material shall pass through 0.15 -0.212 
mm IS sieve  ISO 8397  
vi. Moisture, % by weight, 
max. ( solid carrier -based)  30% - 40 %  AOAC 965.08  
vii. N-fixation  Not less than 10 mg  N/g of sucrose  Annex L  
viii. Shelf life   Should have at least 6 months for sol id 
carrier -based inoculants and 6 weeks for 
liquid -based inoculants (under 
refrigeration) from date of manufacturing   
 
5   Contaminants  
 
Heavy metal contamination in Bio -fertilizers shall not exceed the limits given in table 5 when determined by 
the metho ds prescribed in AOAC 2006.03.  
 
 
Table 5 — Heavy metal contaminants limits  
 
SL No.  Parameter  Limits in ppm  Test methods  
    
i) Arsenic, As, max.  10.0  
    
ii) Cadmium, Cd, max.  5.0 
AOAC 2006.03     
iii) Mercury, Hg, max.  0.1  
    
iv) Chromium, Cr, max.  50.0  
    
v) Lead, Pb, max.  30.0  
    
 
 
6 Sampling  
 
6.1 Scale of sampling  
 
6.1.1 Lot  
 
All containers in a consignment belonging to the same batch of manufacture shall constitute a lot. If the 
consignment is declared to consist of different batches of manufacture, containers of the same batch shall be 
grouped together and each group so formed shall constitute a separate lot.  
 
Sample shall be tested from each lot for ascertaining conformity to the requirements of this standard.  DEAS 1176:2023  
 
   
6.1.2  Sample size  
 
The number of containers to be selected from a lot for testing for microbiological and other requirements shall 
depend on the size of the lot and shall be in accordance with Table 6.  
 
 
Table 6 – Number of containers to be selected for sampling  
 
No. of containers in t he lot  No. of containers to be selected (n)  
Microbiological  Other tests  
up to 1300  12 18 
1301 to 3200  18 24 
3201 and above  24 30 
 
6.1.3 Sampling method   
The containers to be selected for testing shall be chosen at random from the lot by the following  procedure. 
Starting from any container, count them as 1,2,3…... up to r. Every rth containers thus counted shall be 
withdrawn, r being the integral part of N/n, where N is the total number of containers in the lot and n is the total 
number of containers  to be chosen (Table 6).   
 
6.2     Test samples and reference samples  
 
6.2.1   Samples for microbiological tests  
 
The sample containers selected for microbiological tests (see col. 2 of Table 6) shall be divided at random into 
three equal sets and labelled with a ll particulars of sampling. One of these sets of sample containers shall be 
for the buyer; another for the supplier and the third set is the reference. The sample for testing should be 
handled as per prescribed conditions on the manufacturer label.  
 
 
6.2.2  Samples for other tests   
The sample containers selected for other tests (see col. 3 of Table 6) shall be divided at random into three 
equal sets and labelled with all the particulars of the sample. One of these sets of sample containers shall be 
for the b uyer, another for the supplier and third is the reference.  
 
6.2.3   Reference samples  
 
Reference samples shall consist of set of sample containers for microbiological tests (see 6.2.1) and a set of 
sample containers for other tests (see 6.2.2) and shall bear the  seals of the buyer and supplier or as agreed 
to between the two.  
 
 
7 Packaging  
 
Biofertilizers shall be packaged in the materials that are clean and non -defective that protects the product 
from physical, chemical and moisture contamination and withstand m ultiple stages of handling (transportation 
and storage).  DEAS 1176:2023  
4 © EAC 2023  – All rights reserved  
  
8. Labelling  
 
The following information shall be clearly labelled on the package legibly and indelibly in either Kiswahili or 
Kiswahili and English;  
 
a) name of the product i.e. “Biofertilizer”  
 
b) active ingredient shall appear in close proximity to the name of the product by specifying the genus 
and species of the microorganism;  
 
c) brand name;  
 
d) microbial density;  
 
e) name and address of manufacturer, exporter, packer and/or dispatcher;  
 
f) type/nature of c arrier;  
 
g) batch or code number;  
 
h) storage conditions and instructions;  
 
i) date of manufacture;  
 
j) expiry date;  
 
k) net content by weight;  
 
l) product registration number;  
 
m) directions/instructions for use including crops and how to apply  
 
n) safety precautions;  
 
o) Directions on Disposal  
 
p) Compatibility  
 
q) Declaration on GMO status  
 
 
 
 
 
 
 
 
 
 DEAS 1176:2023  
 
  Annex A  
(informative)  
Procedures for quality control of biofertilizer  
A1 Rhizobium  
Quality checks on rhizobium biofertilizer can be divided into three parts:  
a) Mother culture test  
b) Broth test  
c) Peat test  
A1.1 Mother culture test  
Before producing rhizobium bio fertilizer, the mother culture should be checked on the following:  
a) Growth  
b) Purity  
c) Gram strain  
a) Growth  
By streaking a mother culture on yeast mannitol+Congo red agar  (YMA) plates, checking the growth of 
rhizobia.  
Fast-Growing rhizobia colonies will appear in 3 -5 days and slow -growing rhizobia will appear in 5 -7 days.  
b) Purity  
Check purity by streaking culture on glucose peptone agar plate, and incubate for 24 hours a t 30 ºC. No 
growth or poor growth should be obtained on GPA. Good growth and colour changes can be expected from 
contaminants.  
c) Gram stain  
A lot of mother culture is checked by gram staining. Rhizobial cell is Gram -negative, retains safranin colour. 
Cells should appear red and not violet when observed under the microscope.  
A1.2 Broth test  
The following qualities of the broth samples shall be checked to make sure that the broths are in good 
condition.  
a) pH  
b) Staining  
c) Optical density  
d) Total count  
e) Viable number  
 DEAS 1176:2023  
6 © EAC 2023  – All rights reserved  
 a) pH 
Slow -growing rhizobia such as rhizobia for soybeans, mungbean and peanut produce a little basic compound.  
After incubation, the pH will increase (example, pH before growing = 6.0. after growing pH = 6.1 -6.2) If broth 
pH decreases, it m eans some contaminants occur; lower pH indicates presence of contaminants.  
b) Staining (Gram stain or fushin stain)  
Rhizobial cells are stained for observation of shape and size of the cells. Cells of rhizobia are rod -shaped, with 
one or two cells sticking  together. They do not appear in long -chain. Long -chained cells are indicative of 
contaminants.  
Gram - stained cells should appear red, not violet. Fuchsin staining is an easier and faster method. Rhizobial 
cells can be routinely checked using fuscshin stai n. 
Table 1 — Gram stain  
Solutions  Reactions and appearance of bacteria  
Gram -positive  Gram -negative  
I Crystal violet (CV)  Cells stain violet  Cells stain violet  
II Iodine solution  CV-I formed within cells; Cells 
remain violet  CV-I formed within cells; Ce lls 
remain violet  
III Alcohol  Cell walls dehydrated,  
Shrinkage of pores occurs,  
Permeability decreases,  
CV-I complex cannot pass out 
of cells,  
cells remain violet  Lipoid extracted from cell walls,  
porosity increases,  
CV-I is removed from cell  
IV Safranin Cells not affected, remain violet  Cells take up this stain, become 
red. 
 
c) Optical density  
Broth culture with active rhizobial growth will become turbid in 3 -4 days. Broth turbidity, or optical density 
using spectrophotometer (at 540 nm) will show r eadings of 0 -1.0 O.D. The value of O.D correlates to number 
of cells. If values are high, then cells numbers are also high.  
d) Total count  
Total count includes viable cells and dead cells by using petroft -Hauser counter. At least 10 small squares all 
aroun d the total area are counted, and not only in one large square.  
Precautions:  
a) cells have to be homogenous;  
b) clumping of cells (use non -ionic detergent);  DEAS 1176:2023  
 
  c) it gives total count only;  
d) Petroft, cover slip shall be properly positioned to get uniform de pth. 
e) Viable count (To check if its only informative or test method)  
The number of living cells is counted by spread plate or drop plate methods. Doing spread plate by making 
serial dilutions from 10-1 -10-6 or 10-7 (depend on concentration) then three r eplicates of 0.1 mL of broth from 
10-6 and 10-5 are spread over the YMA+CR plates. Plates are incubated (at 28 ºC for seven days. Colonies of 
rhizobial cells are round, opaque and have smooth margin. They are white and do not absorb red colour as 
well as o ther bacteria. Calculation of the number of rhizobia per mL:  
No. of cells /mL = No. of colonies dilution factor  
                               Volume of inoculum  
A1.3 Peat test  
For the peat inoculants, these are the qualities to be checked:  
a) pH  
b) Moistu re content  
c) Viable number  
d) Plant infection method (MPN)  
a) pH (check if it a test method or an informative)  
Maintain neutral pH for the inoculant. Since peat is acidic the pH is to be increased with CaCO3.Weigh 10 g of 
inoculant, pour 20 ml of distille d water, mix well with glass rod, incubate at least 30 minutes, and then 
measure with pH meter.  
b) Moisture content  
The optimum moisture content of peat -inoculant is between 40 % - 50 %. At low moisture rhizobia will die 
rapidly. If moisture is high, inocul ants may stick to the plastic bag and, thus, not good for rhizobial growth.  
c) Viable number  
The number of viable rhizobia is counted by spread -plate method as in the broth test. It is more difficult when 
analyzing non -sterile peat. Colonies may sometimes be contaminated by other bacteria. Good expertise is 
required to conduct this microbiological analysis.  
d) Plant infection method  
Principle  
This is an indirect method of assessing plant infection on nodulation. It is widely used when peat is not sterile. 
It takes more time than spread plate method (because plants have to be grown). MPN is usually done to 
compare the results with a spread plate method.  
Assumptions  
i) If a viable rhizobium is inoculated on its specific host, nodules will develop on those root s. 
ii) Nodulation on that inoculated plant is a proof of the presence of infective rhizobia.  
iii) Absence of nodule is a proof of the absence of infective rhizobia.  DEAS 1176:2023  
8 © EAC 2023  – All rights reserved  
 iv) Uninoculated plants are used as control, with absence of nodule.  
Conditions  
i) Nodulati on on that inoculated plant is a proof of the presence of infective rhizobia. (Nodules however shall 
have pink nodules if cut open hence showing effective bacteria present).  
ii) Absence of nodule is a proof of the absence of infective rhizobia or presence of white (meaning 
noninfective) or grey/green (dead) bacteria in the nodules.  
Estimation of MPN  
Plants within any given pouch are considered as a growth unit. Nodulation is recorded positive for ‘nodulated 
growth unit’ or negative for absence of nodule. Th e actual number of nodules on each plant has no meaning 
on MPN count. If replications are in quadruplicated, the reading may be 4,3,2,1 or 0 units. The highest dilution 
should show no nodulation.  
The estimated number rhizobium per g is calculated by the fo rmula:  
X =  
  
 
Where  
M is the number from MPN,  
d is the lowest dilution (first unit),  
V is the volume of aliquot inoculated.  
Contaminants have some effect on counting. In the presence of contaminants; count of MPN will give lower 
results than p late counts.  
 
A2 Non - symbiotic nitrogen fixer  
In the laboratory, microbial growth may be represented by increase in cell mass, cell number or any cell 
constituent. Utilization of nutrients or increase in metabolic products can also be related to the growt h of the 
organism.  
Growth, therefore, can be determined by several techniques based on one of the following measurements:  
a) Cell count directly by microscopy or by an electronic particle counter, or indirectly by colony count.  
b) Cell mass, directly by w eighing or measurement of cell nitrogen, or indirectly by turbidity.  
c) Cell activity, indirectly by relating the degree of bio chemical activity to the size of the population.  
 
A2.1 Procedure for bacterial cells count by hemocytometer  
If the formulation i s tested only for bacterial cell counting which will not form colonies on agar medium , it can 
be tested directly in h emocytometer by adopting the Standard Serial Dilution Method. The total bacterial cells 
can be observed under microscope  DEAS 1176:2023  
 
   
A2.1.1 Requiremen ts 
Sample 0.1 mL (Bacterial suspension - either from activated sample or dire ctly from the formulation) and 
hemocytometer (Counting chamber)  
 
A2.1.2 Procedure  
Place a drop of product suspension made from liquid culture (Filtered through 1 or more layer of muslin cloth) 
on the engraved grid and let the preparation stand for 1 to 2 minutes to allow the bacterial cells to settle at the 
bottom.  
Put the cover glass over the grid carefully so that no air bubble enters between the slide and cover glass.  
Slide th e cover glass backwards and forwards until colored rings are visible as the two surfaces of the cover 
glass and slide into close contact.  
Count the bacterial cells in the middle square (E), which consists of 25 groups of 16 small squares, each 
group 0.2 m m squares.  
 
A2.1.3 Results  
Calculate the number of spores per mL of the suspension mathematically as follows:  
No of cells per mL of the product = 
  
 
Where,  
D is the dilution factor  
X is the total number of polyhedral counted  
N is the Number of Squares counted  
K is the Volume above one small squares in cm3  
 
 
A3 Mycorrhiza -the Arbuscular micorrhizal fungi, (AMF)  
Quality control in the production of AMF inoculums is essential for product consistency, reliability and 
reproducibility. This is applied  to the labor atory, preparation room, growth room, and storage room and green 
houses, taking care into the design to achieve the most efficient control in inoculums production.  
 
A3.1 Laboratory quality control  
i) Spores are extracted from selected batches  of mono specific spore cultures in the preparation room.  DEAS 1176:2023  
10 © EAC 2023  – All rights reserved  
 ii) The spores are transported in Petri dishes to the laboratory and placed in the refrigerator before 
examination.  
iii) The Petri dishes are examined under stereoscopic microscopes.  
iv) Description  of the spores from each Petri dish is recorded.  
v) Petri dishes are then cleaned and dried.  
 
A3.2 Preparation room quality control  
i) This room has to be isolated from the green house and growth room and should not receive unsterilized  
soil or potting med ia samples  
ii) Stored materials (cultures; sterilized growth media) are clearly labelled and placed in specific containers  
iii) Floor should always be clean, avoiding sweeping which encourages distribution of dust  
iv) Benches and other surfaces are cleaned  with wet towels.  
v) Containers are surface -sterilized with 10% sodium hypochlorite.  
 
A3.3 Growth room quality control  
i) The growth room should be temperature controlled (22 ºC) and air is exhausted to the outside (no recycling 
of stale air);  
ii) Bench to ps should be painted with anti -microbial paint;  
iii) All surfaces should be sterilized periodically e.g. monthly;  
iv) All samples are checked for contaminants and pathogens;  
v) Watering is done manually with great care to avoid cross -contamination.  
 
A3.4 S torage room quality control  
i) All samples stored are placed in plastic bags, with proper labelling and surface of bags should be wiped 
clean before storage.  
ii) Floors and bench tops are wiped regularly, and dusting or sweeping should be avoided to preven t 
generation of dust.  
 
A3.5 Most probable number (MPN) — Method for estimating number of VAM fungi infective  
propagules in VAM products  
A3.5.1 Introduction  DEAS 1176:2023  
 
  The MPN technique gives a more realistic estimate of the number of infective propagules of VAM fungi  in 
VAM products. It is very useful for VAM fungi because  
i) viable propagules include spores and hyphae (soil, roots),  
ii) spores of some VAM fungi may be difficult to extract,  
iii) some VAM fungi produce only a few infective spores, and  
iv) VAM fungi cou ld complete their life -cycle without sporulating.  
The major advantage of MPN technique is that it enumerates all propagules which have the ability to infect at 
a given point of time.  
 
A3.5.2 Requirements  
Twenty pots (70 cm3), 40 presoacked seeds, sterilize d sand: soil (1:1) mixed soil dilutions, polythene bags (30 
cm x 20 cm) and physical balance.  
 
A.3.5.3 Procedure  
Take 30 g of test VAM product in a polythene bag. Add 270 g of sterilized sand: soil (1:1) mix. Shake  
thoroughly (50 - 75 shakes) to get 10 -1 dilution.  
Make a ten -fold series of VAM product dilutions with the test soil up to 10 -1 dilution (go for higher dilution if 
needed)  
Place diluted VAM product in pots (5 pots per dilution)  
Two seeds of selected host are sown in each pot  
Allow the plants to g row in the polyhouse or growth chamber for 30 days after germination.  
Wash, clear and stain the entire root systems with trypan blue and examine microscopically. Record the 
presence or absence of colonization for each root system separately.  
For each of th e 5 replicates in each of the four dilutions (100, 10 -1, 10 -2 and 10 -3), one might obtain a 
combination of numbers as shown below:  
Dilutions  0 1/10 1/100  1/1000  
No. of plants colonized  5 5 3 2 
 
This means that all the 5 replicate pots are positive for VA M colonization in dilutions 10-1 and 10-2, three are 
positive pots in dilution 10-3 and 2 positive pots in dilution 10-4. For the calculation of MPN of propagules only 
three numbers of the given combination are required as follows:  
 
 N1                                               N2                                     N3 
 10-1                                                                    10-2                                                     10-3 
 5                                                  3                                       2  DEAS 1176:2023  
12 © EAC 2023  – All rights reserved  
  
The first number (N1) is that corresponding to least concentrated dilution in which all (or the greatest number 
of) the tubes are positive for AM colonization. The two other numbers (N2 and N3) are those correspon ding to 
the next two higher dilutions.  
The most probable number of AM propagules can then be calculated using MPN Table. Making use of the 
table with the values of N1, N2 and N3, the value given for the combination 5 3 2 is 1.4. This is the MPN Index 
value . 
To obtain the MPN of infective propagules of VAM fungi in the sample MPN Index value has to be multiplied 
by the middle dilution factor ie. 1.4 x 103 = 140 propagules cm3 of VAM product.  
 
A3.6 VAM fungi root infectivity test  
Roots are removed from the so il and washed gently with tap water and then collected on a 0.5 -mm mesh that 
removes soil particles and retains the small root fragments in order to determine the mycorrhizal status of 
each species in different plots. The roots are fixed in FAA (formalin, glacial acetic acid, 95 % ethyl alcohol, 
and water 2: l:10:7 v/v/v/v), digested at 90 ºC for 1 h. in 7 % KOH solution, and stained in 0.05% trypan blue 
(500 mL glycerol, 50mL 1% HCl, 0.5 g trypan blue, and 450 -mL water) for 10 -15 min at 90 ºC; this is a sl ight 
modification of the Phillips and Hayman (1970) method. After staining, excess stain is removed, and the roots 
are destained in clear acidified glycerine (500 mL glycerol, 450 mL water, and 50 mL 1% HCl). Roots are 
washed and cut into 1 -cm segments. 30  to 40 root segments are randomly selected, placed on a glass slide, 
covered with a cover slip, and examined with a microscope to detect the presence or absence of colonization 
by VAM fungi. Only those segments containing mycorrhizal hyphae and either vesi cles or arbuscules are 
counted as colonized. The number of root segment colonized is expressed as a percentage of total root 
segments in the sample.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 DEAS 1176:2023  
 
  Annex B  
(informative)  
Biofertilizers medium composition  
B.1 Rhizobium  
 Medium composition  
 K2HPO 4 - 0.5 g  
 MgSO 4,7H 2O - 0.2 g  
 NaCl - 0.1 g  
 Mannitol - 10.0 g  
 Yeast extract - 1.0 g  
 Agar - 15.0 g  
 Distilled water - 1 litre  
 1 per cent aqueous Congo red 2.5 mL  
 Adjust the pH at 7 ± 0.2 with N/10 HCl and N/10 NaCl  
 Autoclave at 15 psi pressure and 121°C for 30 min  
 
B.2 Azotobacter  
Medium Composition - (Jensen’s medium)  
 Sucrose - 20.0 g  
 K2HPO 4 - 1.0 g  
 MgSO 4.7H 2O - 0.5 g  
 NaCl - 0.5 g  
 FeSO 4 - 0.1 g  
 CaCO 3 - 2.0 g  
 Agar - 15.0 g  
 Distilled water - 1 litre  
 Adjust the pH at 7.  
 Autoclave at 15 psi and 121°C for 30 minu tes. 
 
B.3 Azospirillum:  
Medium composition (Okon’s medium)  
 K2HPO 4 - 6.0 g  
 K2HPO4 - 4.0 g  
 Distilled water - 500 mL  
 MgSO 4 - 0.2 g  
 NaCl - 0.1 g  
 CaCl 2 - 0.02 g  
 NH 4Cl - 1.0 g  
 Malic acid - 5.0 g  
 NaOH - 3.0 g  
 Yeast extract - 0.05 g  
 Na2MoO4 - 0.002 g  
 MnSO4 - 0.000 1 g 
 H3BO3 - 0.0014 g  
 Cu(NO3)2 - 0.0004 g  
 ZnSO4 - 0.0021 g  
 FeCl3 - 0.002 g  
 Distilled water - 500 mL  
 Bromothymol blue - 2 mL  DEAS 1176:2023  
14 © EAC 2023  – All rights reserved  
  (0.5 per cent alcoholic solution)  
 
B.4 Phosphobacter  
Medium composition (Pikovskaya’s medium)  
 Glucose - 10.0 g  
 Ca3(PO4)2 - 5.0 g  
 (NH4 )2SO4 - 0.5 g  
 KCl - 0.2 g  
 MgSO4.7H2O - 0.1 g  
 MnSO4 - trace  
 FeSO4 - trace  
 Yeast extract - 0.5 g  
 Agar - 15.0 g  
 Distilled water - 1 litre  
 Sterilize the medium at 10 psi pressure for 30 minutes  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 DEAS 1176:2023  
 
   
 
 
 
 
 
Annex C  
(informativ e) 
Properties of a good carrier material for seed inoculation  
a) non -toxic to inoculants bacteria strain  
b) good moisture absorption capacity  
c) easy to process and free of lump -forming materials  
d) easy to sterilize by autoclaving or gamma -irradiation.  
e) available in adequate amounts  
f) inexpensive  
g) good adhesion to seeds  
h) good pH buffering capacity  
i) non -toxic to plant  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 DEAS 1176:2023  
16 © EAC 2023  – All rights reserved  
  
 
 
Annex D  
(informative)  
Common carriers for biofertilizers  
SL/NO  Carrier material  Inoculum bacterium  Characteristic  
i. Sterilized oxalic acid 
industrial waste  Rhizobium  -seed inoculation  
-Rhizobium multiplication in  
carrier in ambient 
temperature  
up to 90 days.  
ii. Alginate -perlite dry 
granule  Rhizobium  -soil inoculation  
-the inoculants can be stored  
in a dry state w ithout losing  
much viability.  
iii. Composted sawdust  Bradyrhizobium, rhizobium 
and Azospirillum  -seed inoculation  
-good growth and survival of  
inoculants strains  
iv. Mineral soils  Rhizobium  -seed inoculant  
v. coal Rhizobium  -seed inoculant  
vi. Soybean oil or peanut 
oil added  
with lyophilized cells  Rhizobium  -seed inoculant  
vii. Wheat bran, sugar 
cane baggers  Rhizobium/brady 
rhizobium and rock 
phosphate solubilising 
fungus Aspergillus niger  -soil inoculant  
viii. Nutrient supplemented 
pumice  Rhizobium  -seed inoculants  DEAS 1176:2023  
 
  Annex E  
(normative)  
 
Determination of number of Rhizobium cells  
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 
C.1.3 The culture medium shall be prepared in accordance with ISO 11133 or using  any other acceptable 
validated test method.  
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.  
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. DEAS 1176:2023  
18 © EAC 2023  – All rights reserved  
 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 
matter, in plates for pinpoint colonies. To distinguish colonies from dirt, specks and other foreign matter,  
examine doubtful objects carefully. 
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. DEAS 1176:2023  
 
   DEAS 1176:2023  
20 © EAC 2023  – All rights reserved  
 Anne x F (normative)  
Test for nodulation (Pot culture test)  
Plant nutrient solution  
Reagents  
 Com position Concen tration g/l 
(1) Potassium chloride 0.001M 0.0745 
(2) Di-Potassium  hydro gen Phosphate (K 2HPO4) 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. 
 
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 is prepar ed in the tap water provided  the water  is soft. 
NOTE 2  The nutrient solution has  to be shaken well to disperse  calcium sulphate before  dispensin g. 
NOTE 3  If the solut ion 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.  DEAS 1176:2023  
 
  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 has to be used  as a surface  
Sterilants  for 20 to 30 min. 
NOTE 2  It is reco mmended that the seeds are 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 
seeds 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 
are 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 s oil. 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). 
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. DEAS 1176:2023  
22 © EAC 2023  – All rights reserved  
 Annex G  
(normative)  
 
Determination of number of phosphate solubilizing bacterial cells  
Preparation of cul ture medium  
A.1.1  Use a medium of the following composition: 
Glucose  10.0 g 
Tri‐calcium phosphate 5.0 g 
Ammoniu m sulphate 0.5 g 
Magnesium sulphate  0.1 g 
Sodium chloride  0.2 g 
Yeast extract 0.5 g 
Mang anese sulphate  Trace 
Ferrous suIphate Trace 
Distilled water  1000 ml 
Agar  15.0 g 
pH adjusted  to 7 ± 0.2 
A.1.2  The culture medium shall be prepared in accordance with ISO 11133 or using any other accepta ble 
validated test meth od. 
Preparation of serial dilution for cell counts  
A.2.1  Dispense 30 g of PSBI  in 270 ml of sterile water  and shake  for 10 minutes on  a reciprocal 
shaker. 
A.2.2 Make  serial dilutions up to 10-7 level. 
A.2.3  Pipette out 0.2 ml aliquots of 10-5 to 10-7 dilution and deliver it on the Petri dishes containing set 
medium as described in A.1. 
A.2.4  Spread the  aliquots ov er the plate. Invert the plates and place them in the incubator at 28 ± 2 C 
for 3 days. 
A.2.5  Use 3 replicates of 10-5, 10-6 and 10-7 dilution. 
Counting  
Count the total number of colonies  on the plates  including colonies with solubiliza tion zone  with the help of 
a colony counter.  
Methods for counting s olubilisation zones  
A.4.1  Take 10 g of PSBI  (BF) in 90 ml in water.  DEAS 1176:2023  
 
  A.4.2  Make  a tenfold dilution series up to 10-7 
A.4.3  Take 0.2 ml aliquots of 10-5 to 10-7 dilution using sterile pipettes and delivered  to Petri dishes 
containing Pikovskayei’s media. 
A.4.4 Spread  it unifor mly. Invert the plate  and incubate them up to 2 weeks at 28 ± 2 C. 
A.4.5  Count  the colonies showing hallow cones and measure their diameter. Minimum  accep table zone 
is 10 mm in diameter.  DEAS 1176:2023  
24 © EAC 2023  – All rights reserved  
 Annex H  
(normative)  
 
Determination of soluble ph osphorus using ascorbic acid  
Principles  
C.1.1  Soluble phosphorus forms hetropoly molybdo ‐phosphate complex with molybdate ions which on 
reduction produces a characteristic blue colour measured at 840 to 880 nm.  
C.1.2  Considering the higher stability of the ascorbic acid, easiness to handle, higher tolerance to the 
concentration of interfering  ions, possibilities to use it with all types of acids and higher stability the developed 
colour (from 10 to 60 min), ascorbic acid instead of stanous chloride is nowadays use as the reducing agent 
for the hetropoly molybdo ‐phosphate complex formed by the soluble phosphate ions on addition of ammonium 
molybdate solution.  
Apparatus  
Spectrophotometer capable of transmission measurem ents at 840 to 880 nm. Extractant: It is olsen extract. 
Reagents  
C.3.1  Ammonium molybdate [(NH 4)6M07024.4H20] 
C.3.2  L‐Ascorbic  Acid  
C.3.3  p‐Nitrophenol  
C.3.4  10 N H2S04 
C.3.5  4 N H2S04 
Preparation of reagents  
Sulphomulybdic acid  
C.4.1.1  Take 20 g of ammonium molybdate and dissolve in 300 ml of distilled water.  
C.4.1.2  Add slowly 450 ml of 10 N H2SO 4. 
C.4.1.3  Cool the above mixture and add 100 ml of 0.5 perce nt solution  of antimony potassium tartrate. 
C.4.1.4  Cool and make the volume to one litre. 
C.4.1.5  Store  in glass bottle  away from direct sunligh t. DEAS 1176:2023  
 
  Preparation of mixed reagents  
C.4.2.1  Add 1.5 g of L‐ascorbic acid in 100 ml of the above  stock solution  and mix. Add 5 ml of this 
solution to develop colour.  
C.4.2.2  Mixed reagent is to be prepared fresh as it does not keep for more than 24 h. 
Procedure  
C.4.3.1  Weigh the required material in a 100 ml conical flask.  
C.4.3.2  Add 50 ml  of extractant and shake it of 30 min on a rotary shaker.  
C.4.3.3  Filter the suspension through Whatman filter paper No. 40. If the filtrate is coloured then add a tea 
spoon of Darco ‐60 (activated phosphorus free carbon), shake and fiIter.  
C.4.3.4  Take a known aliquot (5 to 25 ml) of the extract in a 50 ml volumetric flask.  
C.4.3.5  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 4NH 2SO 4. The yellow colour will disappear when the pH of the 
solution becomes 3. Swirl gently to avoid loss of the solution along with the evolution of CO4.(CO 2.) 
C.4.3.6  When the CO 2 evolution has subsided, wash down the n eck of the flask and dilute the solution to 
about 40 ml.  
C.4.3.7  Add 5 ml of the sulphomolybdic acid mixed reagent containing ascorbic acid. Swirl the content 
and make up the volume.  
C.4.3.8  Measure the transmission after 30 min at 880 nm using red filter.  The blue colour developed 
remains stable up to 60 minutes.  
C.4.3.9  Record the concentration of phosphorus (P) in the extract from the standard curve.  
Calculation  
Calculate the concentration  of soluble Phosphor us using the following formula: 
P (%) = 
10050 1050z-6

xy  
where,  
x Weight of the substance taken (g) 
y Volume of the extract  taken for P determ ination (ml) 
z Reading from the standard curve (ppm)  against percent transmission recorded 
Volume of the extractant  added = 50 ml DEAS 1176:2023  
26 © EAC 2023  – All rights reserved  
 Volume made after colour developed = 50 ml 
Preparation of standard curve  
Prepare 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-l i n e  relationship between the soluble P and percent T when plotted  on a semi‐log graph 
paper. DEAS 1176:2023  
 
  Annex I  
(normative)  
 
Determination of the number of Azospirillum cells  
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 
 
Preparation of serial dilution for MPN count  
G.2.1  Dispense 30 g of Azospirillum inoculants in 270 ml of sterile water and shake for 10 minutes o n a 
reciprocal shaker. Make serial dilutions up to 10 -7 dilution.  
G.2.2  Pipette out 0.2 ml aliquots of 10 -4 to 10 -8 dilution and deliver it to screw cap tubes or petridishes 
containing set medium as described in E ‐2.1. Spread the aliquots over the plates. Invert 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.  
Counting  
G.3.1  Count 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.  
G.3.2  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 coun t with MPN table to get the number of cells per gram of the carrier.  DEAS 1176:2023  
28 © EAC 2023  – All rights reserved  
 Azos pirillum count/g  of carrier = 
product of massDry level Dilution  table MPN from Value 
 DEAS 1176:2023  
 
  Annex J  
(normative)  
 
Test for effective root growth  
Pot Culture Test  
H.1.1  For the purpose of 'Pot Culture Test' plant nutrition s olution, 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, seeds 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 f rom 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 may be concluded that the culture is of required quality.  
Method fo r estimating MPN count for Azospirillum  
H.2.1  Add 1 00 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 ‐surface while particles (+ or ‐) in malate semi ‐solid medium, a change to 
dark blue colour of the medium.  
Calculation  
H.3.1  To calculate the most probable number of organisms in the original sample, select as P1 the number 
of positive tubes in the least concentrated dilution in which all tubes are positive or in which the greatest 
number of tubes is +ve, and let P2 and P3 represent the numb ers of positive tubes in the next two higher 
dilution.  DEAS 1176:2023  
30 © EAC 2023  – All rights reserved  
 H.3.2  Then find the row of numbers in Table E.1 in which P1 and P2 correspond to the values observed 
experimentally. Follow that row of numbers across the table to the column headed by the observed val ue of P.  
H.3.3  The figure at the point of intersection is the most probable number of organisms in the quantity of 
original sample represented in the inoculum added in the second dilution. Multiply this figure by the 
appropriate dilution factor to obtain t he MPN value.  
Tables H.1 — Most Probable Numbers for use with 10 Fold dilutions and S Tubes per dilution 
(Cochran, 1950)  
 DEAS 1176:2023  
 
  Annex K  
(normative)  
 
Total plate count of Azotobacter  
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 nutr ient 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 c ulture medium shall be prepared in accordance with ISO 11133 or using any other 
acceptab le validated test method.  
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. 
Incubation of plates  
Label the plates  and incubat e at 28±1 C for 4 to 6 days. 
Colony counting aids  
Same as in C.4. (annex E) 
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 DEAS 1176:2023  
32 © EAC 2023  – All rights reserved  
 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. DEAS 1176:2023  
 
  Annex L  
(normative )  
 
Test for nitrogen fixation in pure culture  
Preparation of pure culture medium  
Prepare medium as given in E.1, excluding agar. 
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. Concentrate 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. 
Determination by Kjeldahl method  
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 should 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 40 g commercial potassium sulphides  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 
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. DEAS 1176:2023  
34 © EAC 2023  – All rights reserved  
 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) is 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. 
Apparatus  
F.3.2.1  For digestion  
F.3.2.1.1   Use kj eldahl’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. 
Procedure  
F.3.3.1  Place 0.25 g of the sam ple in the digestion flask. Add 0.7 gm mercuric oxide, 15 gm potassium 
sulphate followed by 25 ml of sulphuric acid. Shake, let stand for about 30 minutes and heat carefully until 
frothing ceases. Boil briskly 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. 
NOTE Check  the ammonia  recording periodically, using inorganic  nitrogen control, for example, ammoni um sulphate. 
Calculation  
Nitrogen c ontent,  (% by mass) = 
m1.4N T.V.  DEAS 1176:2023  
 
  Where;  
  T.V.   titration value (ml of standard acid)  
  N      normality of standard acid, and  
  M        mass of sample taken.  
Determination of sucrose consumed  
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) 
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. 
Determination of reducing sugars  
F.4.3.1  Reagents  
F.4.3.1.1  Copper sulphate solution (Solution A) : Dissolve 34.639 g of copper sulphate crystals 
(CuSO4.5H5O) 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.  
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  DEAS 1176:2023  
36 © EAC 2023  – All rights reserved  
 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. 
Calculation  
Total reducing sugars  
Total redu cing 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  
Determination of sucrose  
.95 (%m/m)] inversion) before sugars (reducing - (%m/m) inversion) after sugars [(reducing  m/m) (% Sucrose  
 DEAS 1176:2023  
 
  Determination of nitrogen fi xed per g of sucrose consumed  
Nitrogen fixed (mg)/g sucrose consumed = 
(F.5.2) consumed Sucrose(F.3.4) (%m/m) content Nitrogen  DEAS 1176:2023  
38 © EAC 2023  – All rights reserved  
 Bibliography  
 
 EAS 456, Organic production  standard.  
 
 US 1576,  Bio fertilizers — Specification  
 
 KS 2356,  Bio fertilizers — Specification  
 
 WD TC020 -07-2023    
 
© EAC 2023  – All rights reserved