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methods.
Key steps/objectives
1)
2)
3)
4)
5)
6)
7)
8)
9)
10)
(a)
Preparing inoculants
(Key step 1)
Allow the
(b)
(Key step 2)
They are
As in
Leonard jars and growth tubes are also frequently used for MPN
counts.
As in authentication (Exercise
Growth
in Appendix 8.
(c)
To prevent the growing radical from pushing the seed out of the
pouch, a hole is made in the trough of the wick and the radical
is inserted into the hole during planting.
in the trough with fine tipped, sterile forceps when the wick is
wet.
Two forceps are needed: one for holding the wick the other
Figure 5.1.
When the plants are 5-7 days old, reorganize the growth pouches
on the rack.
plants.
(d)
Make
Similarly inoculate the plants which have been set up for the MPN
count.
from the highest dilution and proceed down the series with the
same pipette.
Make the final observation after 3 weeks and record presence (+)
(e)
For each set, write down the dilutions used and record the
nodulation.
If
Each series
The MPN is calculated from the most likely number (m) found in
the MPN tables.
l)
2)
3)
4)
5)
6)
The MPN may now be calculated from "m" by using the following
formula:
m =
d =
v =
Table 5.1.
DILUTION
NUMBER OF
NODULATED
UNITS
II
III
IV
10-2
10-3
10-4
10-5
10-6
10-7
10-8
10-9
0
Total
18
Requirements
(a)
Preparing inoculants
(c)
Incubator
Pipettes, 10 ml sterile; pipettes, 1 ml sterile
Pasteur pipettes, calibrated, sterile; rubber bulbs for
Pasteur pipettes
Flame; spray bottle with alcohol
Erlenmeyer flasks of 2 liter capacity (four) containing 900
ml sterile water each
Rubber stoppers, sterile, to fit 2 liter flasks
Dilution tubes with 9 ml sterile water; racks
Plant nutrient solution
YMA containing Congo Red (YMA-CR)
YMA containing Brilliant Green (YMA-BG)
Plants in growth pouches from (b)
Peat inoculant from (a)
(e)
Records of observations
MPN tables (Appendix 14)