REAGENT PREPARATION
ESTIMATION OF TOTAL ORGANIC CARBON
The determination of organic carbon in the samples was carried out as per the
procedure of Walkley and Black (1974) method.
PRINCIPLE
Organic carbon present in organic matter is oxidized by chromic acid in the
presence of concentrated sulphuric acid.
Potassium dichromate in reaction with
sulphuric acid provides nascent oxygen which combines with carbon and carbon
dioxide.
The sulphuric acid enables easy digestion of organic matter by rendering heat of
dilution. Only a certain quantity of chromic acid is used for oxidation. The excess
chromic acid is left unused by the organic matter with 0.5N ferrous sulphate or ferrous
ammonium sulphate using diphenylamine indicator.
REAGENTS
1N potassium dichromate, Dipheylamine indicator, 0.5N ferrous sulphate or
ferrous ammonium sulphate, concentrated sulphuric acid and phosphoric acid,
(b)
Procedure
Exactly 0.05g of each sample (passed through 0.2 mm sieve) was weighed and
transferred separately to 500ml conical flasks. To the sample 10 ml of 1N potassium
dichromate was added and mixed well by swirling the flask. Then, 20 ml of
concentrated sulphuric acid was added and mixed by gentle rotation for one minute to
ensure complete contact of the reagent with the sample. The contents were allowed to
stand for 20-30 minutes. The flasks were kept on an asbestos sheet to avoid burning of
table due to intense heat. After 30 minutes, 200 ml of water, 10 ml of phosphoric acid
and 1 ml of diphenylamine indicator were added and titrated with ferrous ammonium
sulphate. The dull green colour at the beginning shifted to a turbid blue as the titration
proceeds.
At the end point the colour sharply changed to bright green colour.
Simultaneously, a blank titration (without sample) was run and the volume of 0.5 N
ferrous ammonium sulphate consumed for the blank was noted down.
Calculation:
Weight of sample taken
Volume of 1N potassium dichromate used
-
0.05g
10ml
Volume of 0.5 N ferrous ammonium sulphate used for blank titration -
κ ml
Volume of 0.05 N ferrous ammonium sulphate used for sample titration - y ml
κ ml of sulphuric acid reduces 10 ml of 1 N potassium di-chromate. Therefore, y ml of
sulphuric acid reduces y/x ml.Hence, actual quantity of 1N potassium dichromate used
for oxidation of organic matter - 10-(10xy/x) ml.
2)
Estimation of total nitrogen (N)
The total nitrogen content of the sample was estimated by the Micro-kjeldahl
method (Tandon 1993). This method involved two steps. (i) digestion of the sample to
convert the N compound in the sample to the NH4+ form and (ii) distillation and
determination of NH4 in the digest.
(i)
Digestion of the sample
(a)
Reagents
(i)
Sulphuric-salicylic acid mixture (1.0 g of salicylic acid to 30 ml of con. H2SO4)
(ii)
Sodium thiosulphate and
(iii)
Sulphate mixture: 20 parts of K2SO4 + 1 part of catalyst mixture (20 parts of
CuSO4 + 1 part of selenium powder).
b)
Procedure
To a 100 ml Kjeldahl flask 0.5 g of dried sample was transferred. 20 ml of the
sulphuric-salicylic acid mixture was added and swirled gently so as to bring the dry
sample in contact with the reagents. It was allowed to stand overnight. Next day 5 g of
sodium thiosulphate was added and heated gently for about 5 minutes. Care should be
taken to avoid frothing. The contents were cooled; 10 g of sulphate mixture was added
and digested on Kjeldahl apparatus at full heat for 1 h. When the digestion was
completed, the digest was cooled, diluted, and distilled as described below:
(ii)
Distillation by Kjeldahl method
To vaccum jacket of micro-kjeldahl distillation apparatus, 10 ml of the digest
was transferred.
In a conical flask, 10 ml of 4% boric acid solution was taken
containing bromocresol green and methyl red indicators, to which the condenser outlet
of the flask was dipped. After adding the aliquot digest, the funnel of the apparatus was
washed with 2-3 ml of de ionized water and 10 ml of 40% NaOH solution was added.
Five ml aliquot was distilled to the flask containing 10 ml of boric acid.
After
completion of distillation, the boric acid was titrated against N/200 H2SO4. Blank was
also carried out to the same end point as that of sample.
(iii)
Calculation
The total nitrogen content of the sample was calculated as follows:
Weight of sample = 0.5 g; Normality of H2SO4 = N/200
Volume of digestion = 100 ml; aliquot taken = 5ml
Titrevalue (TV) = SampleTV - BlankTV
N % =
TV × 0.00007 × 100×100
(or) N (%)  TV  0.28
1 ml of N/10 H 2SO4 = 0.00014 g N 
ESTIMATION OF TOTAL NITROGEN (N)
The total nitrogen content of the sample was estimated by the Micro-kjeldahl
method (Tandon 1993). This method involved two steps. (i) Digestion of the sample to
convert the N compound in the sample to the NH4+ form and (ii) distillation and
determination of NH4 in the digest.
(1)
Digestion of the Sample
(a)
Reagents (i) Sulphuric-salicylic acid mixture (1.0 g of salicylic acid to 30 ml of
con. H2SO4) (ii) Sodium thiosulphate and (iii) Sulphate mixture: 20 parts of K2SO4 + 1
part of catalyst mixture (20 parts of CuSO4 + 1 part of selenium powder).
b)
Procedure
To a 100 ml Kjeldahl flask 0.5 g of dried sample was transferred. 20 ml of the
sulphuric-salicylic acid mixture was added and swirled gently so as to bring the dry
sample in contact with the reagents. It was allowed to stand overnight. Next day 5 g of
sodium thiosulphate was added and heated gently for about 5 minutes. Care should be
taken to avoid frothing. The contents were cooled; 10g of sulphate mixture was added
and digested on Kjeldahl apparatus at full heat for 1 h. When the digestion was
completed, the digest was cooled, diluted and distilled as described below:
(2)
Distillation by Kjeldahl method
To vaccum jacket of micro-kjeldahl distillation apparatus, 10 ml of the digest
was transferred.
In a conical flask, 10 ml of 4% boric acid solution was taken
containing bromocresol green and methyl red indicators, to which the condenser outlet
of the flask was dipped. After adding the aliquot digest, the funnel of the apparatus was
washed with 2-3 ml of deionized water and 10 ml of 40% NaOH solution was added.
Five ml aliquot was distilled to the flask containing 10 ml of boric acid.
After
completion of distillation, the boric acid was titrated against N/200 H2SO4. Blank was
also carried out to the same end point as that of sample.
(3)
Calculation
The total nitrogen content of the sample was calculated as follows:
Weight of sample = 0.5 g; Normality of H2SO4 = N/200
Volume of digestion = 100 ml; aliquot taken = 5ml
Titre value (TV) = sample TV – Blank TV
N % =
TV  0.00007  100  100
1 ml of N / 10 H2SO4 = 0.00014 g N 
(Or) N (%) = TV × 0.28
ESTIMATION OF TOTAL PHOSPHORUS
1.
Background
The Olsen test is recommended for calcareous soils with a pH of greater than
5.5. It should be noted that for more acidic soils (pH less than 5.5) this test will likely
overestimate phosphorus content. The Olsen (Bicarbonate) phosphorus test has a
reproducibility of ±10% and a minimum detection limit of 2 ppm.
2.
Reagents
2.1)
Olsen Extracting Fluid (0.5N NaHCO3, pH 8.5)
2.1.1) Accurately weigh (to the nearest 0.1mg) 42.0g of reagent grade sodium
bicarbonate.
2.1.2) Quantitatively transfer to a 1.0L volumetric flask containing 850mL of de
ionized water.
2.1.3) Place on magnetic stirrer until sodium bicarbonate is completely
dissolved.
2.1.5) Adjust pH to 8.5 with a 50% (w/w) sodium hydroxide solution.
2.1.6) Bring total volume to 1L with de ionized water.
2.2)
Acid Molybadate Stock Solution
2.2.1) First prepare a 5M sulphuric acid solution by adding 74ml of
concentrated H2SO4 to 400 ml of de ionized water. Bring to a total
volume of 500 ml. Set this solution aside, it will be used later.
2.2.2) Accurately weigh (to the nearest 0.1mg) 6.0g of ammonium molybdate.
Dissolve in 125ml of de ionized water. Set this solution aside, it will also
be used later.
2.2.3) Accurately weigh (to the nearest 0.1mg) 0.1455g of antimony potassium
tartrate. Dissolve in 50mL of de ionized water.
2.2.4) In a 1.0L volumetric flask, add the anti money potassium tartrate solution
and the ammonium molybdate solution prepared in Step 2.2.2 to the
500mL 5M sulphuric acid prepared in step 2.2.1.
2.2.5) Bring this solution to a total volume of 1.0L with de-ionized water. Mix
well and store solution in fridge.
2.3)
Olsen Color Development Reagent (Prepare same day as analysis)
2.3.1) Accurately weigh (to the nearest 0.1mg) 0.5278g of ascorbic acid.
2.3.2) Dissolve in 100mL of the Acid Molybdate Solution prepared section 2.2.
2.3.3) Store at room temperature, this solution will not keep longer than 24
hours.
2.4)
Stock Phosphorus Standard Solution (50 ppm P)
2.4.1) Dry ~0.5g of monobasic potassium phosphate (KH2 PO4) in oven at
100°C for 15 min.
2.4.2) Cool in desiccator.
2.4.3) Accurately weigh (to the nearest 0.1mg) 0.2197g of the dried KH2 PO4
2.4.4) Dissolve in 50 ml of deionized water.
2.4.5) Bring total volume to 1.0L with distilled water. This standard is stable for
6 months in fridge.
3)
Standards and Calibration Curve
3.1) Using the Stock Phosphorus Standard Solution prepared in section 2.4 and
table 3.1 below, prepare working standard solutions for a calibration curve. Use Olsen
Extracting Fluid to dilute the working standards.
3.2) Accurately pipette 5 mL of each working standard into a 125mL
Erlenmeyer flask.
3.3) Add 15 mL of deionized water.
3.4) Add 5 ml of Olsen Colour Development Reagent. Swirl to mix.
3.5) Allow 10 minutes for colour development.
3.6) Within 2 hours, read the % transmittance at 882 nm using HP 8452A UV
Visible Spectrophotometer.
3.7)
Prepare a calibration curve, by plotting known concentration vs. %
transmittance.
4)
Analysis Procedure
4.1) Homogenize soil by shaking or using a grinder. Avoid non representative
matter such as sticks or rocks.
4.2) Accurately measure 1.5 2 g of soil, and qualitatively transfer to a 125 ml
Erlenmeyer flask.
4.3) Add 40 ml of Olsen Extracting Fluid to the flask. Swirl to mix.
4.3) Secure on mechanical shaker. Cap flasks with rubber stoppers or plastic
wrap to prevent spilling.
4.4) Shake flasks on high for 30 minutes.
4.5) After 30 minutes, remove flasks from shaker and filter through Whatman
#2 filter paper. Refilter if solution is not clear.
4.6) Accurately transfer 5 ml aliquot to a new 125mL Erlenmeyer flask. Dilute
with 15 ml of distilled water.
4.7) Add 5 ml of Olsen Colour Development Reagent to the flask. Swirl to
mix.
4.8) Allow 10 minutes for colour development.
4.9) Within 2 hours, measure the transmittance of solution at 882 nm using HP
8452A UV Visible Spectrophotometer.
4.10) Using the calibration curve developed in section 3, determine the
phosphorus concentration in the filtrate.
4.11) Determine the soil phosphorus concentration according to calculations in
the the following section.
5)
Calculation
5.1) Calculate the Phosphorus Concentration in the Soil (ppm) using the
following
equation:
Phosphorus Concentration in Soil (ppm) = Cfiltrate × 20
Cfiltrate = Phosphorus Concentration in Filtrate (ppm)
5.2) Calculate the Phosphorus Concentration in the Soil (lbs/acre) using the
following equation:
Phosphorus Concentration in Soil (lbs/acre) = Cfiltrate × 40
Cfiltrate = Phosphorus Concentration in Filtrate (ppm)
ANALYSIS OF METAL IONS – DTPA EXTRACTION METHOD
Heavy metals analysis was done according to AOAC (1995) for non-volatile
heavy metal. For this, 1.0 g powder of each sample was digested in HNO3 and HClO4
(9:1) using the wet digestion method by heating slowly on a hot plate in the fume hood
chamber until a clear solution was obtained. The final volume of the solution was made
up to 25 ml with de ionized water. All necessary precautions were adopted to avoid
possible contamination of the samples. Analysis was done using atomic absorption
spectrophotometer (AA 6300, Shimadzu, Japan). The standard reference material of all
the metals (Merck, USA) was used for calibration and quality assurance for each
analytical batch. The efficiency of digestion of samples was determined by adding
standard reference material of metals to different samples. After addition of standards,
samples were digested, and metals were estimated as described above. Three replicates
were analyzed to assess precision of the analytical techniques, and results were
averaged.
DTPA extraction procedure
A 50.0-g (ODW to the nearest 0.001 g) subsample of the wet, un oxidized
sample is weighed into a 500-mL polycarbonate centrifuge bottle and centrifuged at
4°C and 9,500 rpm for 30 min. The supernatant is decanted; pH is determined on the
supernatant and represents the saturated sediment pH. To the sediment remaining in the
centrifuge bottle is added 250 ml of 0.005 M DTPA + 0.01 M calcium chloride + 0.1
M tri ethanolamine solution buffered at pH 7.3. The bottle is sealed, placed on a
mechanical shaker, and centrifuged as before. The supernatant is carefully poured into a
polyethylene bottled and analyzed for metals according to the methods described in
USEPA (1986).
For the diethylene triaminepentaacetic acid–triethanolamine triethanolamine
extraction (DTPA–TEA), compost samples of 1:5 (sample:extractant w/v) were shaken
at 200 rpm for 2 h and centrifuged at 8000g for 5 min. After filtration, the supernatants
were stored in polyethylene bottles before analysis.
Sequential extraction
The conventional method of Tessier et al. (1979) was used for the sequential
extraction. The extraction was carried out in polypropylene centrifuge tubes of 50 ml
capacity with an initial mass of 2.5 g oven dried (105°C) fine fraction (<1 mm) of the
samples.
The following steps were adopted:
(1) Exchangeable: 2.5 g of sample was extracted with 25 ml of 1 M MgCl2 at pH
7.0 with agitation at 220 rpm for 1 h at 25 °C.
(2) Acid extractable: Residue from (1) was extracted with 25 ml of 1M NaOAc at
pH 5.0 with agitation for 5 h at 25 °C.
(3) Reducible: Residue from (2) was extracted with 25 ml of 0.1 M (NH2OH. HCl)
in 25% acetic acid (v/v) for 6 h at 96 °C in a water bath with agitation.
(4) Oxidizable: To the residue from (3), 5 ml of 30%, H2O2 at pH 2.0 was added at
30 min interval to revent foaming (Gupta and Chen, 1975). The mixture was
heated to 85 °C for 5 h, and after cooling, ml of 3 M NH4OAc in 20% HNO3
(v/v) was added with continuous agitation.
(5) Residual: Residue from (4) was digested with HF and HClO4 (5:1) for 2 h at
120°C.
After each successive extraction, the supernatant was collected after centrifugation
at 8000g for 5 min and filtered through a 0.45 lm membrane filter and diluted to
volume. The residue was washed with 10 ml of Milli-Q water by shaking and
centrifugation without loss of solids. Total metal content from a separate sample was
analysed to evaluate the performance of sequential extraction by digesting it with HF
and HClO4 (5:1) (Tessier et al., 1979). Extractions were performed in triplicates and the
mean values are presented with standard deviation. Heavy metal concentrations of all
extracts were determined spectrometrically by Perkin Elmer AA-6300, double beam
atomic absorption spectrophotometer (AAS) and graphite furnace AAS.
MICROBIOLOGICAL ANALYSIS
At periodic intervals of 15 days the total bacterial load in the vermibed
substrates were counted. The methodology was based on the 'Standard Dilution Plate
Technique' where three random sub-samples were collected. 1 gm of composite sample
was weighed and then transferred to flasks containing 10 ml of sterile deionized water.
The flasks were then shaken for half an hour at 120 rpm on rotary shaker. From this
original dilution, the serial dilutions were prepared and 0.1 ml of the selected dilution
was used for plating on nutrient agar medium for further identification.
Enumeration of total colony forming units of microorganisms
The total colony forming units (CFU) of bacteria in the vermibed materials at
the beginning of the experiment (initial) and at the end of the experiment (wormworked and worm-unworked substrates) were enumerated using standard plate count
method (Subba Rao, 1995; Kannan, 1996).
Initial, final compost and the dissected gut contents of earthworm, and were
added into separate flasks containing 99 ml of sterile distilled water blanks. Further
serial dilutions were made using 9 ml sterile distilled blanks.
After serially diluting the sample 0.1ml of the aliquots of the appropriate
dilutions were spread plated onto the sterile nutrient agar plates for bacteria, Rose
Bengal agar for fungi and Ken knight’s Media for actinomycetes. The plates were
incubated for 48 - 72 hours at 37˚C.
The petriplates with 30-300 colonies were selected for enumeration bacterial
population. The bacterial, fungal and actinomycetes population was expressed as
colony forming units (CFU) per gram of the sample.
One gram of each sample was taken in a sterile conical flask containing 9ml of
distilled water and shaken in a vortex mixer for 30 minutes. From this stock, various
dilutions were prepared from 10-1 to 10-7 with sterile distilled water. One ml of the
diluted sample was poured into petriplates containing media for bacteria.
Three
replicates were maintained for each observation. The initial microbial CFU and the
final microbial CFU (worm-worked and worm-un worked) in the vermibed substrates
were subjected to one-way ANOVA using the Computer Software, SPSS (Version
11.1).
Identification of bacteria
Isolation, characterization, and identification of bacterial and fungal species in
the gut region of earthworm, and in vermicompost of different wastes were done using
cultural, biochemical and specific characteristics by standard microbiological
procedures. The gut region of earthworm was divided into three portions viz., foregut,
midgut, and hindgut in specific lengths and used for studying the micro flora.
After noting the morphology and pigmentation of the colony, the
morphologically dissimilar colonies were randomly selected and the pure culture was
maintained on nutrient agar and stored at 4˚C for identification of the isolates. The
organism grown over the above medium was analyzed for their morphological, cultural
and biochemical characters.
At intervals, colony-forming units were determined by making total counts on
the incubated plates using a colony counter. Representative colonies emerging from the
plates were grouped according to their cultural characteristics, purified by repeated subculturing and maintained on appropriate agar slants as stock cultures. The bacterial
isolates were tested for Gram reaction (Claus, 1992), morphology, motility, catalase
and oxidase reactions, citrate utilization, coagulase production, starch hydrolysis and
sugar fermentation (Harrigan and McCance, 1976, Seeley and Van Demark, 1972). The
isolates were then identified with reference to Sneath et al. (1986).
The generic composition of the bacterial strains were identified by following the
methods of Shewan et al., (1960), Gerhardt, et al., (1981), Starr et al., (1981) and
Bergey’s Manual of Determinative Bacteriology (1989).
Morphological studies
The isolate was subjected to the Gram’s staining technique by Claus (1992).
Gram’s staining
Differential staining as per Gram’s method is based on the fact that one group of
microorganisms (Gram - positive),
after treatment with the stain
form the
tripheylmethane group and mordanting or differentiating with Lysol’s solution, no
longer gives up the colour on alcohol treatment where as the other group of bacteria
(Gram- negative) are decolorized ion alcohol. Gram positive bacteria are stained
bluish – black and Gram negative bacteria are stained red.
Procedure
A thin smear was prepared over a glass slide heat fixed and air dried. The heat
fixed smear was flooded with crystal violet for one minute and washed with distilled
water. Mordanting was done by adding lugol's iodine for the period of one minute as
per the above step. Decolorizing was done by using 96% alcohol or acetone or acetone
iodine solution, till the disappearance of colour of the previous stain in the follow of the
decolorizer. Counter staining was done with safranine for one minute washed well and
the stained smears are air dried and observed by oil immersion objective (100x).
Motility Test
The motility bacteria are occasionally evident from the large colonies of the
microbes on solid culture media, but the active motility was verified systematically by
investigation by hanging drop.
Here a drop of a liquid culture or bacterial suspension in sterile tap water was
dropped on a cover glass. A hollow ground slide, with a cavity bordered with Vaseline,
was superimposed and quickly inverted. The edge of the drop was focused under a
microscope with low magnification, in the first instance and the intrinsic motion of the
bacteria is examined thereafter with the oil immersion objective.
BIO-CHEMICAL TESTS
1)
IMViC Test
Indole test: By adding tryptone to the nutrient medium, the capacity of the bacteria to
decompose the tryptophan is verified by means of p-methylaminobenzaldehyde in an
acid solution. The 24 hour broth culture will show red colour ring at the top if it was
positive.
Methyl red reaction: The colony from the plate was inoculated with MR-VP broth
tubes and incubated at 37°C for 24 hours. The positive result was indicated by the
formation of red colour after the addition of methyl red, with a pH value of 4.4
(transition point of methyl red) is reached by the bacterial hydrolysis of glucose.
Voges-proskauer reaction: Acetyl methyl carbinol (Acetoin) formed as an
intermediate product of carbohydrate decomposition in MR-VP broth which contains
glucose can be detected by the formation of pink to bright red colour on the addition of
Barrit's reagent to the medium indicates positive reaction.
Citrate utilization test: Slants of Simmon's citrate agar medium was inoculated with
the organism (grown on the plate) and incubated (with the organism grown on the plate
and incubated) at 37°C for 24 hours. Change of the colour from green to blue indicates
the positive reaction.
2)
Amino acid Hydrolysis Test: The amino acid hydrolysis medium containing
Bromocresol purple as indicator was inoculated by stabbing and incubated at 35°C for
48-72 hours and change from purple to yellow colour indicates positive results.
3)
Catalase Test: Hydrogen peroxide (3%) was added to 24 hours nutrient broth
culture. The presence of effervescence in the medium indicates the positive result.
4)
Carbohydrate Fermentation Test: The test culture was inoculated to the
sterilized O/F medium in tubes. A duplicate of the above mentioned O/F medium was
prepared with addition of a layer of sterilized petroleum jelly to a depth of 5-10mm.
The inoculated tubes were incubated at 35°C for 3 days. Acid production in aerobic
condition indicated an oxidative breakdown of sugar and acid production throughout
the tubes with anaerobic condition indicated as fermentative breakdown of sugar.
5)
Oxidase test: The filter paper impregnated with teteramethyl-p-phenyl-diamine
dihydrochloride reagent was streaked with overnight culture of the test organism, the
change of colour to purple was recorded as positive.
6)
Sugar utilization test: Sterile phenol red broth containing appropriate sugar
with inverted Durham's tube was inoculated with the test culture. The change of colour
from red to yellow and upliftment of the Durham's tube shows the acid and gas
production which indicates positive results.
7) Triple Sugar Iron Agar (TSI): To the prepared sterile Triple sugar iron agar slants
the test sample was stabbed and incubated at 35°C for 24 hours. The acid production
was indicated by colour change from red to yellow and H2S production by blackening
of medium
8) Urease test: The urea agar slant was inoculated with a heavy inoculum and
incubated at 35°C for 18-24 hours. The change of colour from yellow to red was
recorded as positive. Selective isolation medium and their specific growth
characteristics are observed by inoculation on appropriate medium. For the
identification of the bacterial isolates, many suitable media and reagents were used. The
composition of the media and reagents were given
9)
Lipids hydrolysis: lipids are the complex mixture of fatty acids and glycerol
linked together by ester bonds. They possess high molecular and also act as reservoirs
of large amount of energy. Their degradation therefore requires the enzymatic activity
of lipase with out which their entry through the microbial cell membrane would be
impossible. However the presence of lipids-splitting enzymes enables their entry after
which they undergo a serious of metabolic activities, either to acquire, provide or
synthesize new protoplasmic compounds. In the experimental procedure tributyrin agar
serves as a supplement for a lipid source. In combination with agar, tributyrin forms an
emulsion. Following the incubation period, a clear zone around microbial growth
confirmed the presence of lipase.
10)
Gelatine
hydrolysis:
gelatine
gains
more
significance
in
bacterial
identification. It is an incomplete protein existin in two forms which change in
temperature , ie, temperature less then 25°c,it exist in gel state whereas at temperature
greater than 25°c it is in liquid state. Microorganisms possessing gelatinase enzymes in
hydrolysis gelatine to low molecualar weight soluble amino acids. Under these
conditions even at a low temperature 4°c, the gelatine cannot maintain its gel state and
it is this liquefaction property that he indicates the presence gelatinase enzyme.
11)
Nitrate reduction test: Some microorganisms still continue to reduce either to
ammonia (NH3+) or to molecular nitrogen (N2). Nitrate reduction process can be
determine by supplementing 0.1% potassium nitrate on a semi solid agar medium.
Since the denitrification requires anoxic condition, o2 penetration is prevented by semi
solid medium. After incubation period, addition of sulphuric acid and alpha napthyl
amine gives a characteristic cherry red colour, a positive result for nitrate reduction.
However, the colour change indicate the conversion nitrate to nitrite and the colourless
cultures therefore have a possibility of getting reduced to ammonia and sometimes even
to molecular nitrogen. To confirm this, zinc powder is added. The presence of red
colour indicates the conversion nitrates to nitrites, a positive result. In the absence of
colour change, it can be conformed that nitrates were reduced to ammonia or nitrogen
gas.
12)
Hydrogen sulphide test:
Utilization of organic sulphur
Breakdown of proteins by photolytic microorganisms leads to the leaves of
amino acids. de gradation of sulphur containing amino acids requires the present of an
enzyme called cysteine desulphurase. Those microorganisms that produce this enzyme
can only utilize the sulphur containing amino acids. These enzymes act on the sulphur
component and reduced it to sulphide.
Utilization of Inorganic sulphur
Certain microorganisms can utilize inorganic sulphur compound such as
thiosulphate (s2o3) and sulphates (so4-) and reduce them to sulphides (so3) and hydrogen
sulphides. The process involves oxidation and the sulphur atom of the inorganic
compound act as hydrogen acceptor.
Since the process requires anoxic condition, semisolid agar medium is used.
The medium supplemented with peptone, sodium thiosulphate and ferrous ammonium
sulphate (indicator). The black precipitate formed along the growth of culture reveals
the evolution of H2s. The precipitate (coloured) is obtained by the complex reaction
between hydrogen sulphide and ferrous ammonium sulphate.
13)
Starch hydrolysis: starch is composed of several molecules of glucose linked
together by glycosidic bonds. It is mixture of amylase (linear form and amylopectin
(highly branched form).being complex in nature, starch needs the amylase enzymatic
activity for hydrolysis. Amylase acts on 1-4 glycosidic linkage of starch and hydrolyses
them in to fragments of dextrin’s and then to maltose molecules. Further hydrolysis to
soluble glucose requires the presence of another enzymes maltase. The concentration
maltose increases at every stage. In this experiment, starch agar is used as supplement
for carbon source. Only the microorganisms with amylase activity can utilize the
substrate and grow. Following the incubation period, then the Petri plate is flooded with
iodine appearance of blue-black colour denotes the presence of insoluble starch
whereas the formation of clear zone indicates microbial growth, hydrolysing the starch
by starch- splitting enzymes