Chapter-5

MATERIAL & METHODS

Present investigation was conducted in department of Biotechnology, R.B.S.

Engineering and Technical Campus Bichpuri , Agra. The details of materials and methods used
during the course of present investigations are being described as under – Fertilizer control
order 1985 Ref. 29

A. METHOD OF ISOLATION OF AZOTOBACTOR BACTERIA

1. PROM sample
2. Apparatus
 Pipettes Graduated 1 ml and 10 ml.
 LAF
 Dilution Bottles or flasks
 Petri Dishes Clear, Uniform, flat-bottomed.
 Hot Air Oven
 Autoclave
 Incubator
 Hand Tally or Mechanical counting Device
 pH meter
3. Reagents:
Congo red
Medium Use a plating medium of the following composition
Contains In/ltr
Agar 20g

Sucrose (C12 H 22O11) 20.0 g

Ferric sulphate Fe2 (SO4) 0.1g

Dibasic potassium phosphate (K 2HPO4) 1.0g

Magnesium sulphate (MgSO4, 7H 2O) 0.5g

Sodium Chloride (NaCl) 0.5g

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 Calcium carbonate (CaCO3) 2.0g

Sodium Molybdate (Na 2MoO4 ) 0.005gms

Distilled water 1000ml

pH 6.8 to 7.2
Sterilizing and preparation procedure for plates:
Sterilize the sampling and plating equipment with dry heat in a hot air oven at not less
than 160o C for not less than 2 hours.
 Sterilize the media by autoclaving at 120oC for 20 min. To permit passage of steam
into and from closed container when autoclaved, keep stoppers slightly loosened or
plugged with cotton. Air from with in the chamber of the sterilizer should be ejected
allowing steam pressure to rise. Preparation of Plating Medium and Pouring
 Prepare growth medium in accordance with the composition of the specific Bacteria.
 Melt the required amount of medium in boiling water or by exposure to flowing steam
in partially closed container but avoid prolonged exposure to unnecessarily high
temperature during and after melting. Melt enough medium which will be used with in
3h. Re-sterilization of the medium may cause partial precipitation of ingredients.
 When holding time is less than 30 min promptly cool the molten medium to about
45oC, and store until used, in a water bath or incubator at 43oC to 45oC.
Pouring
Introduce 12 to15 ml of liquefied medium or appropriate quantity depending on size of
the Petri dish at 42 to 44o C into each plate. Gently lift the cover of the dish just
enough to pour in the medium. Sterlize the lips of the medium container by exposure to
flame.
a. Immediately before pouring.
b. Periodically during pouring,
c. When pouring is completed for each batch of plates, if portion of molten
medium remain in containers and are to be used without subsequent
sterilization for pouring additional plates. As each plate is poured thoroughly
mix the medium with test portions in the Petri dish.

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  By rotating and tilting the dish and without splashing the medium over edge, spread
the medium evenly over the bottom of the plate. Provide conditions so that the medium
solidifies with reasonable promptness (5-10min) before removing the plates from
surface.
4. Incubation of plates
a. Label the plates and incubate at 28±2o C for 4 to 6 days.
b. Identify bacteria by congo red ,or gram staining method
Count the colonies with the aid of magnifying lens under uniform and properly controlled,
artificial illumination. Use a colony counter, equipped with a guide plate and rules in
Centimeter Square. Record the total number of colonies with the hand tally. Avoid mistaking
particles of undissolved medium or precipitated matter, in plates for pinpoint colonies. To
distinguish colonies from dirt, specks and other foreign matter, examine doubtful objects
carefully.
Morphology of Azotobacter
Int.J.Curr.Microbiol.App.Sci (2015), Ref. 30
Azotobacter is shaped cells cocoid, oxidase negative, catalase positive and form cysts, which
serves to protect against extreme environmental conditions, such as drought, ultraviolet light
and ionizing radiation (Madigan et al., 1997). Azotobacter is a cocci bacterium with sized 1.5
2.0 m, not forming endospores but cyst. Move with flagella, are aerobic and chemo
organotroph, using glucose, alcohol, salt of organic material to grow, catalase positive. The
optimum pH at 7-7.5 and commonly found in soil and water. Certain species can be associated
with the roots of plants (Holt, et al., 1999). Shape colony isolates obtained from generally
spherical and irregular, varied colonies such as wavy edges, parted, intact and curly. The most
dominant elevation colonies on bacterial isolates are flat and the other curved and flat arise.
Colors of bacteria are white and yellow, while the rounded shape and stem cells. The results of
gram reaction test showed that all isolates are Gram negative.
RESULT:-
The result of related research is explaind by following method & discussion which are as
following
Azotobactor:-
Protocol:-

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1. Sterilized all required glassware
2. Prepair planting agar media and sterilized in oven for 15 psi or 30 minutes
3. Pour media in petri plates
4. Prepair fertilizer solution for azotobactor isolation ,dilute 30 gm to 270 ml distriald
water
5. Prepare a standard solution and ,10-1, 10-2,10-3,10-4 serial dilution solution
6. Inoculate dilution solution in each perti plate
7. Incubate petri plate in incubator for 4-7 days on 28±30c

34
Glassware Washing Media Prepration

Pouring Sample Preparation

35
 Serial dilution solution Inoculation

Discussion :-
In the petri plate we see the white/milky white, round, colonys of azotobactor which is
identify by congo red and gram staining method.

Azotobactor standard Inoculation 10-1 Inoculation

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10-2 Inoculation 10-3 Inoculation

10-4 Inoculation

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Identification
1. Azotobector Indentified By congo red
Method
prepair 1% congo red solution
take 2-2.5 ml solution and put in petri plate and put for 30 second ,then discart extra solution
from petri plate ,wash petri plate by water and observe
Result:- It was observed by the formation of white/ milky white round that azotobactor
bacteria is present.

Azotobector colony identified by Congo red Indicator

2. Azotobector Indentified by Gram Staining Method

Required material
Slide, loop, water, cristal violet, iodine ,95% alcohol, sefrin indicator
Method:-
1. Put a drop of water on slide
2. Take bacteria colony on slide
3. Spread the colony on slide and dry slide by sprit lamp
4. Put 2-3 drop Cristal voilet and wait for 30 second
5. Wash slide by water
6. Put 2-3 drop iodine on slide and wait for 30 second

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 7. Wash slide by water
8. Put 2-3 drop 95% alcohol on slide
9. Wash slide by water
10. Put 2-4 drop sefrinin on slide and wait 30 second
Result:-
We can see that azotobactor is a gram negative Bacteria.

Azotobactor gram staning slide A Azotobactor gram staning slide B

B. Method of Analysis of Phosphate Solubulising Bacterial Bacillus Megatherium

1. Apparatus – Same as azotobactor

2. Reagents
Congo red
3. Medium

Use a plating medium of the following composition:

Contains In/ltr

Glucose 10.0g

Tri-calcium phosphate 5.0 g

Ammonium sulphate 0.5 g

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 Magnesium sulphate 0.1 g

Sodium Chloride 0.2 g

Yeast extracts 0.5 g

Manganese sulphate Trace

Ferrous sulphate Trace

Distilled water 1000ml

Agar 15.0 g

pH adjusted to 7 ± 0.2

4. Sterilizing and preparation procedure for plates:

 Sterilize the sampling and plating equipment with dry heat in a hot air oven at not less
than 160o C for not less than 2 hours.
 Sterilize the media by autoclaving at 120oC for 20 min. To permit passage of steam
into and from closed container when autoclaved, keep stoppers slightly loosened or
plugged with cotton. Air from with in the chamber of the sterilizer should be ejected
allowing steam pressure to rise. Preparation of Plating Medium and Pouring
 Prepare growth medium in accordance with the composition of the specific Bacteria.
 Melt the required amount of medium in boiling water or by exposure to flowing steam
in partially closed container but avoid prolonged exposure to unnecessarily high
temperature during and after melting. Melt enough medium which will be used with in
3h. Re-sterilization of the medium may cause partial precipitation of ingredients.
 When holding time is less than 30 min promptly cool the molten medium to about
45oC, and store until used, in a water bath or incubator at 43oC to 45oC.
Pouring
Introduce 12 to15 ml of liquefied medium or appropriate quantity depending on size of
the Petri dish at 42 to 44 oC into each plate. Gently lift the cover of the dish just enough
to pour in the medium. Sterilize the lips of the medium container by exposure to flame.
a. Immediately before pouring.
b. Periodically during pouring, and

40
 c. When pouring is completed for each batch of plates, if portion of molten
medium remain in containers and are to be used without subsequent
sterilization for pouring additional plates. As each plate is poured thoroughly
mix the medium with test portions in the Petri dish.
 By rotating and tilting the dish and without splashing the medium over edge spread the
medium evenly over the bottom of the plate. Provide conditions so that the medium
solidifies with reasonable promptness (5-10min) before removing the plates from
surface.
5. Incubation of plates
a. Label the plates and incubate at 28±2o C for 4 to 6 days .
b. Colony counting aids
Count the colonies with the aid of magnifying lens under uniform and properly controlled,
artificial illumination. Use a colony counter, equipped with a guide plate and rules in
Centimeter Square. Record the total number of colonies with the hand tally. Avoid mistaking
particles of undissolved medium or precipitated matter, in plates for pinpoint colonies. To
distinguish colonies from dirt, specks and other foreign matter, examine doubtful objects
carefully.
Count all plates but consider for the purpose of calculation plates showing more than 30 and
less than 300 colonies per plate. Disregard colonies, which absorb Congo red and stand out as
reddish colonies. Bacillus Megatherium stands out as white, translucent, glistening and elevated
colonies. Count such colony numbers and calculate figures in terms of per gram, of carrier.
Also check for freedom from contamination at 10-5 dilution.
Morphology of Bacillus Megatherium Bacteria
NEHU bacillus megatherium ref.31
Cell morphology-
 Rod,dimeter 5-7 µm ,cell length -10 µm,Motile, gram positive
Colony morphology –
 circuler, entire, opaque, white
Growth condition-
 temp- 320c optimum,pH-7
 Growth condition-Aerobic, days- 1-2

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RESULT :-
The result of related research is explaind by following method & discussion which are as
following
Bacillus megatherium :-
protocol:-
1. Sterilized all required glassware
2. Prepair planting agar media and sterilized in oven for 15 psi or 30 minutes
3. Pour media in petri plates
4. Prepair fertilizer solution for azotobactor isolation ,dilute 30 gm to 270 ml Distriald
water
5. Prepare a standard solution and ,10-1, 10-2,10-3,10-4 serial dilution solution
6. Inoculate dilution solution in each perti plate
7. Incubate petri plate in incubator for 4-7 days on 28±30c

42
Glassware Washing Media Prepration

Pouring Sample Preparation

43
 Serial dilution solution Inoculation

Discussion:-
In the petri plate we see the white,round,circular colonys of Bacillus Megatherium bacteria
which is identify by congo red and gram staining method.

B. Megatherium Standard 10-1 Inoculation

Inoculation

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10-2 Inoculation 10-3 Inoculation

10-4 Inoculation

45
Identification
1) By congo red indicator
Method :-
prepair 1% congo red solution
take 2-2.5 ml solution and put in petri plate and put for 30 second ,then discard extra solution
from petri plate ,wash petri plate by water and observe
Result:- It was observed by the formation of slight bubbles and round shaped that bacteria is
present.

B. Megatherium Colony Indentified by Congo red Indicator

2) By gram staining
Required material
Slide, loop, water,cristal violet, iodine ,95% alcohol, sefrin Indicator
Method:-
1. Put a drop of water on slide
2. Take bacteria colony on slide
3. Spread the colony on slide and dry slide by sprit lamp
4. Put 2-3 drop Cristal voilet and wait for 30 second

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 5. Wash slide by water
6. Put 2-3 drop iodine on slide and wait for 30 second
7. Wash slide by water
8. Put 2-3 drop 95% alcohol on slide
9. Wash slide by water
10. Put 2-4 drop sefrinin on slide and wait 30 second

Result:-
We can see that Bacillus megatherium is a Gram Positive Bacteria.

B. Megatheriom gram staning slide A B. Megatheriom gram staning slide B

Discussion :-
The study of PROM (phosphate rich organic manure) producing bacteria’s name
Azotobactor or Bacillus Megatherium Is present in the PROM fertilizer we identified those
bacteria’s by congo red and gram staning method.

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