TRAINING REPORT ON BIO-

FETILIZER
AT
CORDET (IFFCO), PHULPUR, PRAYAGRAJ
FROM: 1 OCTOBER 2020 TO 30 OCTOBER 2020
(4 Weeks)

SUBMITTED TO: SUBMITTED BY:

MR. DPS TOMAR MADHUMITA MISHRA

PRINCIPAL PF CORDET 17BSCAGH133
PHULPUR, PRAYAGRAJ
BSc(HONS) AGRICULTURE
SHUATS, PRYAGRAJ
 ABOUT CORDET

COOPERATIVE RURAL DEVELOPMENT TRUST

Activity: Practical Training Programme for farmers.

IFFCO promoted Cooperative Rural Development Trust (CORDET) in the year 1979 to
provide education and training to farmers. The CORDET units are set up at Phulphur,
Kalol and Kandla locations.

CORDET is instrumental in demonstrating various farming system models to increase the

farm income by organizing various trainings and skill development programs for youth
and women. It has demonstrated crop production system, dairy, balanced fertilization,
use of bio-fertilisers, bee-keeping, pisciculture, computer use, screen printing, welding,
tailoring and embroidery, adult education programmes, fruits and vegetables
preservation etc. at Phulpur and Kalol units.

During the year FY 2017-18, CORDET has organizer 306 training programmes benefiting
17,891 farmers including women from various States. CORDET Phulpur and Kalol also
provide free soil testing facilities through their Soil Testing Laboratories to the farmers
and analysed 95,104 soil samples during the year 2017-18. In addition 21 000 soil samples
were also analysed for six micro nutrients.

To increase microbial activities in the soil, CORDET has increased the production
capacity of liquid bio-fertilisers at Kalol Unit from 1.5 lakh litres to 4.75 lakh litres per
annum. The total production of bio fertilizers during 2017-18 was 8.66 lakh litres.

To promote cows of Indian breed, 66,422 litres of cow milk was produced during FY
2017-18 at Phulpur. Neem oil extraction unit with the capacity of 150 MT/year has been
set up at CORDET Phulpur.

Integrated Rural Development Program (IRDP) has been undertaken in 14 villages. A

variety of social and promotional activities like construction of community centres,
drinking water facility, tree plantation, soil testing campaigns, supply of cattle feed.
promotion of vermin-compost, mini-kit distribution (CIP) etc. were undertaken in these
villages_ Around 175 programmes in various field have been organized during FY 2017-
18, which benefitted 15, 272 farmers.
 INTRODUCTION
A bio fertilizer (also bio-fertilizer) is a substance which contains living microorganisms
which, when applied to seeds, plant surfaces, or soil, colonize the rhizosphere or the
interior of the plant and promotes growth by increasing the supply or availability of
primary nutrients to the host plant. Bio-fertilizers add nutrients through the natural
processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth
through the synthesis of growth-promoting substances. Bio fertilizers can be expected to
reduce the use of synthetic fertilizers and pesticides. The microorganisms in bio fertilizers
restore the soil's natural nutrient cycle and build soil organic matter. Through the use of
bio fertilizers, healthy plants can be grown, while enhancing the sustainability and the
health of the soil. Since they play several roles, a preferred scientific term for such
beneficial bacteria is "plant-growth promoting rhizobacteria" (PGPR). Therefore, they
are extremely advantageous in enriching soil fertility and fulfilling plant nutrient
requirements by supplying the organic nutrients through microorganism and their by-
products. Hence, biofertilizers do not contain any chemicals which are harmful to the
living soil.

Biofertilizers provide "eco-friendly" organic agro-input. Biofertilizers such as Rhizobium,

Azotobacter, Azospirilum and blue green algae BGA) have been in use a long time.
Rhizobium inoculant is used for leguminous crops. Azotobacter can be used with crops
like wheat, maize, mustard, cotton, potato and other vegetable crops. Azospirillum
inoculations are recommended mainly for sorghum, millets, maize, sugarcane and wheat.
Blue green algae belonging to a general cyanobacteria genus, Nostoc or Anabaena or
Toiypothrix or Aulosira, fix atmospheric nitrogen and are used as inoculations for paddy
crop grown both under upland and low-land conditions. Anabaena in association with
water fern Azolla contributes nitrogen up to 60 kg/ha/season and also enriches soils with
organic matter. GA) have been in use a long time. Rhizobium inoculant is used for
leguminous crops. Azotobacter can be used with crops like wheat, maize, mustard, cotton,
potato and other vegetable crops. Azospirillum inoculations are recommended mainly for
sorghum, millets, maize, sugarcane and wheat. Blue green algae belonging to a general
cyanobacteria genus, Nostoc or Anabaena or Biofertilizers provide "eco-friendly" organic
agro-input. Biofertilizers such as Rhizobium, Azotobacter, Azospirilium and blue green
algae (BGA) have been in use a long time. Rhizobium inoculant is used for leguminous
crops. Azotobacter can be used with crops like wheat, maize, mustard, cotton, potato and
other vegetable crops. Azospirillum inoculations are recommended mainly for sorghum,
millets, maize, sugarcane and wheat. Blue green algae belonging to a general
cyanobacteria genus, Nostoc or Anabaena or Tolypothrix or Aulosira, fix atmospheric
nitrogen and are used as inoculations for paddy crop grown both under upland and low-
land conditions. Anabaena in association with water fern Azolla contributes nitrogen up
to 60 kg/ha/season and also enriches soils with organic matter.
 ACKNOWLEDGEMENT
This report is based on in plant training which I have completed in
CORDET, IFFCO PHULPUR. This is an outcome of not only our
effort but of under grudging help and guidance of and co-operation,
which we have received from the industry staff and members.
It is my privilege to express our deep sense of gratitude, immense
respect and pleasure to our project, guide Dr.GAUTAM GOSH under
whose guidance, I have able to being complete my training and also
thankful to MR. DPS TOMAR (Principal, cordet) and ANJALI
CHOWDHARI for imbibing in an aptitude for industrial training
through effective guidance and constant encouragement.
I thankful to all those who have directly or indirectly helped me in my
project and took a constructive interest in it. Without their presence it
would have been impossible to gather all this information which are the
backbone of this report.
 Candidate declaration

I Madhumita Mishra (Id: 17BSCAGH1330), student of BSc

agriculture from shuats,Pryagraj,U.P hereby, I presented a in
plant training under the guidance of Dr. Gautam Gosh during
the period of 1 October 2020 to 30 October 2020 at
cordet,phulpur,pryagraj.
I further want to state that this institute had trained and guided
me about the wide range of techniques related with the quality
control of bacterial bio fertilizer and their applications in
different stages of bio fertilizer production.
 CONTENT

1) BIOFERTILIZFR
(2) TYPES OF BIOFERTILIZER
(3) LIQUID BIOFERTILIZER
(4) PHOSPHATE SOLUBLIZING MICROORGANISMS
(4) IMPORTANCE OF BIOFERTILIZER
(5) APPLICATION OF BIOFERTILIZER

2) DECOMPOSER

3) METHODS AND MATERIAL

(1) ISOLATION
(2) IDENTIFICATION
(3) PURIFICATION
(4) MOTHER CULTURE
(5) FERMENTATION
(6) FORMULATION
(7) BOTTLING

4) RESULT AND DISSCUSSION

5) REFERENCES
BIO-FERTILIZER
'Bio' means 'life'. Therefore, by definition biofertilizers are living organisms that enrich the
nutrient quality of the soil. It refers to the use of microbes instead of chemicals to enhance the
nutrition of the soil. As a result, it is also less harmful and does not cause pollution.

Rhizobium, Azotobacter, Azospirillum and blue green algae (BGA) have been traditionally
used as Biofertilizers. Rhizobium inoculant is used for leguminous crops such as pulses.
Azotobacter can be used with crops like wheat, maize, mustard, cotton, potato and other
vegetable crops

TYPES OF BIOFERTILIZER
Nitrogen Fixer
 Bacteria
 Rhizobium
 Azotobacter
 Mycobacterium
 Azospirillum
 Bacillus

 Blue Green algae

 Anabaena
 Nostoc Tolypothrix A
 Nabseopsis

 Azolla
 Azollafiliculoid
 Azollarubra

 Vascular Arbuscular Mycorrhiza (VAM)

 Plant Growth Promoting Rhizobacteria (PGPR)

 Sulphur Solubilizing Microbes (SSB)

Among them, Bacillus subtilis has the strongest resistance, the most functions, the widest
adaptability, and the most stable effect. Bacillus subtilis can produce substances like cell
division factors and plant growth hormones and promote the growth of plants so that plants can
resist pathogenic bacteria.
The hazards of soil salt accumulation in soil are poor in soil structure, poor air permeability,
high bulk density, increased soil temperature, poor aerobic microbial activity, slow nutrient
release, low permeability coefficient. and strong capillary action, leading to further
intensification of surface soil salinization, Soil cold, hard, board phenomenon. Bacillus subtilis
probiotic has the good effect on soil condition and improve the pH condition in soil.

IMPORTANCE OF BJOFERTILIZER
These are means of fixing the nutrient availability in the soil.
 Since a bio-fertilizer is technically living, it can symbiotically associate with plant roots.
Involved microorganisms could readily and safely convert complex organic material
into simple compounds, so that they are easily taken up by the plants. Microorganism
function is in tong duration, causing improvement of the soil fertility. It maintains the
natural habitat of the soil. It increases crop yield by 20- 30%, replaces chemical
nitrogen and phosphorus by 30%, and stimulates plant growth. It can also provide
protection against drought and some soil-borne diseases.
Some important groups of biofertilizers include:
 Azolla-Anabaena symbiosis: Azolia.is a small, eukaryotic, aquatic fern having global
distribution. Prokaryotic blue green algae Anabaena azolla resides in its leaves as a
symbiont. Azolla is an alternative nitrogen source. This association has gained wide
interest because of its potential use as an alternative to chemical fertilizers.
 Rhizobium: Symbiotic nitrogen fixation by Rhizobium with legumes contribute
substantially to total nitrogen fixation. Rhizobium inoculation is a well-known
agronomic practice to ensure adequate nitrogen.

Rhizobium
Rhizobium is a genus of gram negative soil bacteria that fix nitrogen. Rhizobium species form
an endosymbiotic nitrogen-fixing association with roots of legumes and Parasponia.

The bacteria colonize plant cells within root nodules, where they convert atmospheric nitrogen
into ammonia using the enzyme nitrogenase and then provide organic nitrogenous compounds
such as glutamine or ureides to the plant. The plant, in turn, provides the bacteria with organic
compounds made by photosynthesis. This mutually beneficial relationship is true of all of the
rhizobia, of which the genus Rhizobium is a typical example.

Rhizobium inoculant was first made in USA and commercialized by private enterprise in 1930s
and the strange situation at that time has been chronicled by Fred (1932).

Azotobacter
Azotobacter is a free-living nitrogen-fixing oacterium, which is used as a biofertilizer in the
cultivation of most crops. Azotobacter are usually motile, oval, or spherical bacteria, form
thick-walled cysts, and may produce large quantities of capsular slime.

Azotobacter species are free-living, nitrogen-fixing bacteria; in contrast to Rhizobium species,

they normally fix molecular nitrogen from the atmosphere without symbiotic relations with
plants, although some Azotobacter species are associated with plants. Nitrogen fixation is
inhibited in the presence of available nitrogen sources, such as ammonium ions and nitrates.

Azospirillum
Azospirillum, a free-living nitrogen-fixing bacteria closely associated with grasses.
Azospirillum Bacteria is a Gram negative motile bacteria belonging to the order
Rhodospirillales, associated with roots of monocots, including important crops, such as wheat,
corn and rice.

Azospirillum contains 109/gm spores of Azospirillum species. This Azospirillum bacterium

fixes the atmospheric nitrogen and makes it available to plants in non-symbiotic manner that
can replace 50-90% of the nitrogen fertilizer required by plants.

Azospirillum bio fertilizer also secretes some fungicides, enzymes but in minute amount. Use
of Azospirilium bio fertilizer increases the crop production in large scale. We are engaged in
manufacturing and marketing of Bio control age comprise of different types of beneficial
Bacterial, Fungal and Viral cultures. Azospirillium mainly useful for monocot vegetables.

Azospirillum is an eco-friendly liquid biological fertilizer formulation containing bacteria,

Azospirillum which contain large amount of lipid granules, which enters the cortical cells of the
root and fix up atmospheric nitrogen and also produces biologically active substances like
vitamins, nicotinic acid, in dole acetic acid gibberellins etc and helps in better retention of
flowers and enhances the plant growth.

Phosphate Solubilizing Bacteria (PSB)

Phosphate solubilizing bacteria (P58) are beneficial bacteria capable of solubilizing inorganic
phosphorus from insoluble compounds.0.) P-solubilisation ability of rhizosphere
microorganisms is considered to be one of the most important traits associated with plant
phosphate nutrition. It is generally accepted that the mechanism of mineral phosphate
solubilization by PSB strains is associated with the release of low molecular weight organic
acids, through which their hydroxyl and carboxyl groups chelate the cations bound to phosphate
thereby converting it into soluble forms. PSB have been introduced to the Agricultural
community as phosphate Biofertilizer. Phosphorus (P) is one of the major essential
macronutrients for plants and is applied to soil in the form of phosphate fertilizers. However, a
large portion of soluble inorganic phosphate which is applied to the soil as chemical fertilizer is
immobilized rapidly and becomes unavailable to plants. Currently, the main purpose in
managing soil phosphorus is to optimize crop production and minimize P loss from soils. PSB
have attracted the attention of agriculturists as soil inoculums to improve the plant growth and
yield. When PSB is used with rock phosphate, it can save about 50% of the crop requirement of
phosphatic fertilizer. The use of PS8 as inoculants increases P uptake by plants. Simple
inoculation of seeds with PSB gives crop yield responses equivalent to 30 kg P205 /ha or 50
percent of the need for phosphatic fertilizers. PSB can be applied through fertigation or in
hydroponic operations.

e.g. Bacillus subtilis

Microbial fertilizers are more expensive than organic fertilizers because of the addition of
biologically active bacteria! Therefore, the role of strains is the most important for microbial
fertilizers. At present, the main microbial strains on the market are mainly Bacillus subtilis
probiotic, so understanding the role of Bacillus subtilis probiotic is very important.

Inducing plant-producing resistance means that Bacflius subtilis not only inhibits plant
pathogens, but also induces the plant's own resistance mechanism to enhance the plant's
resistance to diseases. What is PGPR? Internationally, the rhizosphere-promoting bacteria that
can promote plant growth in the soil are generally called Plant growth promoting rhizobacteria
(abbreviated as PGPR).

DECOMPOSER
Decomposers are organisms that break down dead or decaying organisms, and in doing so, they
carry out the natural process of decomposition. Like herbivores and predators, decomposers are
terotrophic, meaning that they use organic substrates to get their energy, carbon and nutrients
for growth and development. While the terms decomposer and detritivore are often
interchangeably used, detritivores must ingest and digest dead matter via internal processes
while decomposers can directly absorb nutrients through chemical and biological processes
hence breaking down matter without ingesting it. Thus ,-vertebrates such as earthworms,
woodlice, and sea cucumbers are technically detritivores, not decomposers, since they must
ingest nutrients and are unable to absorb them externally.

Fungi used as a decomposer in IFFCO is Trichoderma

 Bio fertilizers today

Bio fertilizers provide "eco-friendly" organic agro-input. Bio

fertilizers such as Rhizobium, Azotobacter, Azospirilium and
blue green algae (BGA) have been in use a long time.
Rhizobium inoculant is used for leguminous crops. Azotobacter
can be used with crops like wheat, maize, mustard, cotton,
potato and other vegetable crops. Azospirillum inoculations are
recommended mainly for sorghum, millets, maize, sugarcane
and wheat. Blue green algae belonging to a general
cyanobacteria genus, Nostoc or Anabaena or Tolypothrix or
Aulosira, fix atmospheric nitrogen and are used as inoculations
for paddy crop grown both under upland and low-land
conditions. Anabaena in association with water fern Azolla
contributes nitrogen up to 60 kg/ha/season and also enriches
soils with organic matter.[2][3]seaweeds are rich in various
types of mineral elements (potassium, phosphorus, trace
elements etc) hence they are extensively used as manure by
people of coastal districts. Seaweed - manure also helps in
breaking down clays. Fucus is used by Irish people as manure
on a large scale. In tropical countries bottom mud of dried up
ponds which contain abundant blue green algae is regularly
used as manure in fields. The mixture of seaweeds and blue
green algae may serve as ideal fertilizer
 METHODS AND MATERIAL
1) ISOLATION

STREAK PLATE METHOD OF ISOLATION

A microbial culture consisting of two or more species is said to be a mixed culture, whereas a
pure culture contains only a single species. Obtaining isolation of individual species from a
mixed sample is generally the first step in identifying an organism a commonly used isolation
technique is the streak plate. In the streak plate method of isolation, a bacterial sample (always
assumed to be a mixed culture) is streaked over the surface of a plated agar medium. During
streaking, the cell density decreases, eventually leading to individual cells being deposited
separately on the agar surface. Cells that have been sufficiently isolated vrii4 grow into colonies
consisting only of the original cell type. Because some colonies form from individual cells and
others from pairs, chains, or clusters of cells, the term colony-forming unit (CRJ) is a more
correct description of the colony origin. Several patterns are used in streaking an agar plate, the
choice of which depends on the source of inoculum and microbiologist's preference. Although
streak patterns range from simple to more complex, all are designed to separate deposited cells
(CFUs) on the agar surface so individual cells (CFUs) grow into isolated colonies.

The identification process of an unknown microbe relies on obtaining a pure culture of that
organism. The streak plate method produces individual colonies on an agar plate. A portion of
isolated colony then may be transferred to a sterile medium to start a pure culture.

Inoculation of Agar Plates using the Quadrant Streak Method

This inoculation pattern is usually performed as the streak for isolation of two or more bacterial
species in a mixed culture with suspected high cell density.

1. Obtain the sample of mixed culture with a sterile

2. You have two options at this point. Use whichever is more comfortable for you or is required
by your instructor.

a. Leave the sterile agar plate on the table and lift the lid slightly, using it as a shield from
airborne contamination or,
b. Place the plate lid down on the table). Then remove base and hold it in the air on an angle.

3. starting at the edge of the plate lightly drag the loop careful not to cut the agar surface and
forth across the agar surface be careful not to cut the agar surface.
4. Remove the loop and replace the lid.
5 Sterilize your loop as before. It is especially important to flame it from base to tip now
because the loop has lots of bacteria on it.
6. Rotate the plate a little less than 90°.
7. Let the loop cool for a few moments (or your can, touch an open part of the agar), then
perform another streak with the sterile loop beginning at one end of the first streak pattern.
Intersect the first streak only two or three times.
8. Sterilize the loop, then repeat with a third streak beginning in the second streak
9. Sterilize the loop, then perform a fourth streak beginning in the third streak and extending
into the middle of the plate. Be careful not to enter any streaks but the third.
10. Sterilize the loop.
11. Label the plate's base with your name, date and sample inoculated.
12. Incubate the plate in an inverted position for tie assigned time at the appropriate
temperature.

STREAK
PLATE OF
SERRATIA
MARCESCENS
note the
decreasing
density of growth
in the four streak
patterns. On this
plate, isolation is
first obtained in
the fourth streak.
Cells from an
individual colony
may be
transferred to a
sterile medium to
start a pure
culture.

SPREAD PLATE METHOD OF ISOLATION

The spread plate technique is a method of isolation in which a diluted microbial sample is
deposited on an agar plate and spread uniformly across the surface with a glass rod. With a
properly diluted sample, cells (CFUs) will be deposited far enough apart on the agar surface to
grow into individual colonies.

After incubation, a portion of an isolated colony can be transferred to a sterile medium to begin
a pure culture. The spread plate technique also has applications in quantitative microbiology

Following is a description of the spread plate technique. As in the previous exercises, basic
skills are printed in the regular black type and new skills are printed in blue.

Spread Plate Technique

1. Arrange the alcohol beaker, Bunsen burner, and agar plate as shown in Figure 1-29. This
arrangement minimizes the chances of catching the alcohol on fire.
2. Lift the plate's lid and use it as a shield to protect from airborne contamination.
3. Using an appropriate pipette, deposit the designated inoculum volume on the agar surface
from this point, the remainder of steps should be completed within about 15 seconds to prevent
the inoculum from soaking into the agar.
4. Properly dispose of the pipetting instrument used to inoculate the medium, because it is
contaminated each lab has its own specific procedures and your instructor will advise you what
to do.
5. Remove the glass spreading rod from the alcohol and pass it through the flame not leave the
rod in the flame; the combination of the alcohol and brief flaming are sufficient to sterilize it.
Be careful not to drop any flaming alcohol on the work surface. Be especially careful not to
drop flaming alcohol back into the alcohol beaker.
6. After the flame has gone out on the glass rod, lift the lid of the plate and use it as a shield
from airborne contamination. Then touch the rod to the agar surface away from the inoculum to
cool it.
7. To spread the inoculum, hold the plate lid with the base of your thumb and index finger and
use the tip of your thumb and middle finger to rotate the base. At the same time, move the rod
in aback-and-forth motion across the agar surface. After a couple of turns, do one last turn with
the rod next to the plate edge. Alternatively, place the plate on a rotating platform and spread
the inoculum.
8. Remove the rod from the plate and replace the lid.
9. Return the rod to the alcohol in preparation for the next inoculation. There is no need to
flame it again.
10. Label the plate base with your name, date, organism, and any other relevant information.
11. Incubate the plate in an inverted position at the appropriate temperature for the assigned
time. (If you plated a volume of inoculum greater than0.5 mL, wait a few minutes and allow it
to soak in before inverting the plate).

2) IDENTIFICATION
Introduction to Bacterial Identification, Accurate and definitive microorganism identification,
including bacterial identification and pathogen detect essential for correct disease diagnosis,
treatment of Infection and trace back of disease outbreak associated with microbial infections.

Methods Used to Identify Bacteria. Traits that can be valuable aids to identification are
combinations of cell shape and size, gram stain reaction, acid fast reaction, and special
structures including endospores, granules, and capsules.
Bacteria are classified and identified to distinguish one organism from another and to group
similar organisms by criteria of interest to microbiologists or other scientists. The most
important level of this type of classification is the species level.

 Gram Stain: The Gram stain is a differential stain in which a decolourization step occurs
between the applications of two basic stains. The Gram stain has many variations, but
they all work in basically the same way (Figure 3-86). The primary stain is crystal
violet. Iodine is added as a mordant to enhance crystal violet staining by forming a
crystal violet—iodine complex. Decolourization follows and is the most critical step in
the procedure. Gram- negative cells are decolorized by the solution (of variable
composition-generally alcohol or acetone) whereas Gram-positive cells are not. Gram-
negative cells can thus be colorized by the counterstain safranin. Upon successful
completion of a Gram stain, Gram-positive cells appear purple and Gram-negative cells
appear reddish-pink (Figure 3-87). Electron microscopy and other evidence indicate that
the ability to resist decolourization or not is based on the different wall constructions of
 Gram-positive and Gram negative cells. Gram-negative cell walls have a higher lipid
content (because of the outer membrane) and a thinner peptidoglycan layer than Gram-
positive cell walls. The alcohol/acetone in the decolourizer extracts the lipid making the
Gram-negative wall more porous and in capable of retaining the crystal violet—iodine
complex, thereby decolorizing it. The thicker peptidoglycan and greater degree of cross-
linking (because of teichoic acids) trap the crystal violet—iodine complex more
effectively, making the Gram-positive wall less susceptible to decolourization.

GRAM STAIN OF

STAPHYLOCOCCUS (+) AND PROTEUS (—) (X1320) Staphylococcus has a staphylococcal

arrangement, whereas Proteus is a bacillus.

 Acid-Fast Reaction: The presence of mycolic acids in the cell walls of acid fast
organisms is the cytological basis for the acid-fast differential stain. Mycolic'acid is a
waxy substance that gives acid-fast cells a higher affinity for the primary stain and
resistance to decolourization by an acid alcohol solution. A variety of acid-fast staining
procedures are employed, two of which are the Ziehl-Neelsen (ZN) method and the
Kinyoun (K) method.
These differ primarily in that the ZN method uses heat as part of the staining process,
whereas the K method is a "cold" stain, in both protocols the bacterial smear may be
prepared in a drop of serum to help the "slippery" acid-fast cells adhere to the slide. The
two methods provide comparable results. The waxy wall of acid-fast cells repels typical
aqueous stains (As a result, most acid-fast positive organisms are only weakly Gram-
positive.) In the ZN method, the phenolic compound carbolfuchsin is used as the
primary stain because it is lipid-soluble and penetrate the waxy cell wall. Staining by
carbolfuchsin is further enhanced by steam-heating the preparation to the wax and allow
the stain to move into the cell. Acid alcohol is used to decolorize nonacid-fast cells;
acid-fast cells resist this decolourization. A counterstain, such as methylene blue, then is
 applied. Acid-fast cells are reddish-purple; non-acid- fast cells are blue .The Kinyoun
method (Figure 3-98) uses a slightly more lipid-soluble and concentrated carbolfuchsin
as the primary stain. These properties allow the stain to penetrate the acid-fast walls
without the use of heat but make this method slightly less sensitive than the ZN method.
Decolourization with acid alcohol is followed by a contrasting counterstain, such as
brilliant green or methylene blue.
AN ACID-FAST
STAIN USING THE
ZN METHOD (X1000):
Notice how most of the
Mycobacterium phlei
(AM) cells are in
clumps, an unusual state
for most rods. They do
this because their waxy
cell walls make them
sticky. A few individual
cells are visible,
however, and they
clearly are rods. The
Staphylococcus
epidermidis cells (AF)
are also in clumps, but
that is because they
grow as grape-like
clusters. Each cell's
diameter is
approximately 1pm.

 Capsule Stain: Capsules are composed of mucoid poly saccharides or polypeptides that
repel most stains. The capsule stain technique takes advantage of this characteristic by
staining around the cells. Typically an acidic stain such as Congo red or nigrosin, which
stains the background, and a basic stain that colorizes the cell proper, are used. The
capsule remains, and appears as a white halo between the cells and the colour
background this technique begins as a negative stain; cells are spread in a film with an
acidic stain and are no: heat fixed. Heat-fixing causes the cells to shrink, leaving and
artifactual white halo around them that might be interpreted as a capsule. In place of
heat-fixing, cells may be emulsified in a drop of serum to promote their adhering to the
glass slide.

 Endospore Stain: An endospore is a dormant form of the bacterium that allows it to

survive poor environmental conditions. Spores are resistant to heat and chemicals
because of a tough outer covering made of the protein keratin. The keratin also resists
staining, so extreme measures must be taken to stain the spore. In the Schaeffer-Fulton
method, a primary stain of malachite green is forced into the spore by steaming the
bacterial emulsion. Alternatively, malachite green can be left on the slide for 15minutes
or more to stain the spores. Malachite green is water-soluble and has a low affinity for
cellular material, so vegetative cells and spore mother cells can be decolorized with
water and counterstained with safranin. Spores may be located in the middle of the cell
(central), at the end of the cell (terminal), or between the end and middle of the cell
 (subterrnmal) Spores also may be based on shape—either spherical or elliptical (oval)—
and size relative to the cell (i.e.,whether they cause the cell to look swollen or not).

3) PURIFICATION

Pure Culture Technique

A pure culture may originate from a single cell or single organism, in which case the cells are
genetic clones of one another. 3. Introduce bacteria into a growth medium using "aseptic
technique" to prevent contamination.

Enrichment Culture Method.

 Streak Plate Method: This method is used most commonly to isolate pure cultures of
bacteria.
 Pour Plate Method.
 Spread Plate Method
 Serial Dilution Method.
 Single Cell Isolation Methods.
 Enrichment Culture Method.

4) MOTHER CULTURE PREPARATION

The Mother of all Cultures MyCrobz Mother Culture is the base ingredient in all of our
products and services. It is an organic liquid solution of beneficial and effective
microorganisms that has been cultured with organic molasses and medicinal herbs.

5) FERMENTATION
Carbohydrate fermentation is the metabolic process by which an organic molecule acts as an
electron donor (becoming oxidized in the process) and one or more of its organic products act
as the final electron acceptor (FEA). In actuality, the term "carbohydrate fermentation" is used
rather broadly to include hydrolysis of disaccharides prior to the fermentation reaction. Thus, a
"lactose fermenter" is an organism that splits the disaccharide lactose into the monosaccharides
glucose and galactose and then ferments the monosaccharides. In this section you will see the
term "fermenter" frequently. Unless it is expressly used otherwise, this term should be assumed
to include the initial hydrolysis and/or conversion reactions.

Fermentation of glucose begins with the production of pyruvate. Although some organisms use
alternative pathways, most bacteria accomplish this by glycolysis. The end products of pyruvate
fermentation include a variety of organic acids, alcohols, anti-hydrogen or carbon dioxide gas.
The specific end products depend on the specific organism and the substrate fermented. In this
unit you will perform tests using two differential fermentation media—Phenol Red (PR) Broth
and MR-VP Medium. Phenol Red Broth is a general-purpose fermentation medium. Typically,
it contains any one of several carbohydrates (e.g., glucose, lactose, sucrose) and a pH indicator
to detect acid formation. MR-VP broth is a dual-purpose medium that tests an organism's ability
to follow either (or both) of two specific fermentation pathways. The methyl red (MR) test
detects what is called a mixed acid fermentation. The Voges-Proskauer (VP).test identifies
bacteria that are able to produce acetones as part of a 2, 3-butanediol fermentation. See
Appendix A for more information about fermentation.

Phenol Red Broth: Phenol Red (PR) Broth is a differential test medium prepared as a base to
which a carbohydrate is added. Included in the base medium are peptone and the pH indicator
phenol red. Phenol red is yellow below pH 6.8, pink to magenta above pH 7.4, and red in
between.

EQUIPMENTS IN CORDET
FERMENTERS

MICROSCOPE
During preparation the pH is adjusted to approximately7.3 so it appears red. Finally, an inverted
Durham tube is added to each tube as an indicator of gas production. Acid production from
fermentation of the carbohydrate lowers the pH below the neutral range of the indicator and
turns the medium yellow. Deamination of peptone amino acids produces ammonia (NH3),
which raises the pH and turns the broth pink. Gas production, also from fermentation, is
indicated by a bubble or pocket in the Durham tube where the broth has been displaced.

6) FORMULATION
7) BOTTELING

RESULT AND DESCUSSION

There are various product are prepared as a biofertilizer and decomposer

Decomposers are also prepared in CORDET. Trichoderma used as decomposer. These are
mainly use for the leguminous plant.
REFERENCES
https://www.hindawi.comhournalsibmri/2018/6439481
https://www.google.com
https://www.wikipedia.in
Microbiology Laboratory theory and application at el. Michael J. Leboffe and Burton
E.Pierce/2010