Agriculture Reference
In-Depth Information
supernatant is estimated by the chlorostannous-reduced molybdophosphoric acid
blue method (King
1932
; Jackson
1967
). To 10 ml of supernatant, 10 ml
chloromolybdic acid (ammonium molybdate 15 g; distilled water 400 ml and
10 N HCl 400 ml. These materials are mixed slowly with rapid stirring and cooled,
and the volume is made to one litre with distilled water) and 5 drops of
chlorostannous acid (stannous chloride 10 g; concentrated HCl 25 ml; the stock
solution is kept in airtight bottle and one ml of stock solution is mixed in 132 ml of
distilled water at the time of experiment) are added, and the volume is adjusted to
50 ml with distilled water. The absorbance of blue colour developed is read at
600 nm. The amount of P solubilizer is calculated using the calibration curve of
KH
2
PO
4
. The change in pH following TCP solubilization is also recorded. Each
independent experiment should be repeated three times after several subcultures to
ensure the reproducibility of the results. Solubilization index and SE of the bacterial
isolates showing greater solubilization on both solid and liquid media and persis-
tence of PS activity after several subcultures are the criteria for the selection of
efficient PS strains for further studies.
1.5.4 Microbiological and Biochemical Characterization
of PSM
The phosphate solubilizers are identified firstly by microbiological and biochemical
tests. The microbiological tests may include the assessment of colonial morphology
[shape, margin (serrated or smooth)], colour and characteristics such as the secre-
tion of watery or mucoid/gummy substances from colonies, Gram reaction and
shape of microbes. The biochemical reaction may involve indole reaction, citrate
utilization, methyl red test, Voges-Proskauer, catalase, oxidase test, starch, gelatin,
lipid hydrolysis, mannitol salt utilization test and sugar fermentation test, etc. The
resulting characteristics are compared with those given in
Bergey's Manual of
Determinative Bacteriology
(Holt et al.
1994
), and strains are identified to generic
level only.
1.5.4.1 Antibiotic Sensitivity Behaviour of Isolated Cultures
Antibiotic sensitivity behaviour of the isolated P solubilizers is determined using
the antibiotic discs of known potency by disc diffusion method of Bauer (
1966
)in
order to find antibiotic markers for the PSM strains. For this, freshly prepared and
autoclaved nutrient broth is inoculated by isolated bacterial cultures and incubated
for 24 h at 28
2
C. 100
l of overnight grown test culture is taken on nutrient agar
plates and is evenly spread with sterile glass rod spreader. Plates are then mounted
with individual antibiotic (e.g. amoxicillin, chloramphenicol, ciprofloxacin, cloxa-
cillin, nalidixic
μ
acid, nitrofurantoin, norfloxacin, novobiocin, doxycycline
