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3 D e c e m b e r 1962
INTRODUCTION
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B10 M. E. BROWN, S. K. BURLINGHAM, AND R. M. JACKSON
BASIC METHODS
Soils
The foUowing solls were examined for Azotobacter.
1. Rothamsted allotment soil, pH 7.7. H e a v y clay loam, high organic
content, treated with sewage sludge; mixed garden crops for at least a I00
years.
2. Rothamsted Garden Plots soll, p H 6.1. H e a v y clay loam mixed with
flints; mixed arable cropping for the last 5 years.
3. Rothamsted Great Field soil, p H 5.9. H e a v y clay loam under permanent
grass.
4. Waterbeach, Cambs. Fen peat soll, p H 7.3. Arable land under wheat.
5. Broom's Barn Experimental Station, Marl Pit Field, pH 8.2. Light
clay loam mixed with flints; arable land under barley and then sugar beet.
6. Dunholme Experimental Station, Bean Stack Field, pH 7.8. Light
limestone soil under sugar beet.
All soils were passed through a 3-mm sieve before preparing the soil sus-
pensions. Ten-gram samples fresh weight were suspended in sterile distilled
water made up to 100 ml volume in 10-oz serew-capped bottles. Unless
otherwise stated the suspensions were shaken for 10 min on a reciprocating
machine at 200 strokes per minute with a horizontal excursion of 1'/. The
percentage moisture of the fresh soll was determined and final numbers of
Azotobacter are given per g dry weight soil.
Counting methods
1. P l a t e - c o n n t m e t h o d . The plate-count method for estimating num-
bers of Azotobacter in soil was originally described by J e n s e n 6. Es-
sentially it consists of inoculating the dried surface of a nitrogen-deficient
agar medium with a suitably diluted soil suspension, which is spread over the
agar and allowed to dry before closing the petri dish lid and incuhating.
In our experiments aliquot samples (usually 0.5 rel.) of the soil suspension
were inoculated ort to the surface of the agar, previously dried for 1 h at 25 °,
and spread with a platinum wire. The plates were left open in the incubator
until no longer visibly moist, approximately 2 h. Azotobacter, distinguishable
as cream, mucilaginous colonies, 1 to 2 mm diameter was counted after 3 to
5 days; when the plates were left longer than 7 days colonies of other organ-
isms, which might be confused with Azotobacter, developed. Uninoculated
control plates showed no aerial contamination by Azotobaeter.
METHODS FOR COUNTING AZOTOBACTER 311
RESULTS
water with beads and the dispersing agents had no further effect;
but unnecessarily complicated the method.
N u m b e r s of A z o t o b a e t e r p e r g r a m d r y soil on m e d i a w i t h d i f f e r e n t
c a r b o n sourees
Substrate
Soll
Suerose Glucose I Mannitol ] P=0.05
Allotment 3100 3460 1240 / 1621
G a r d e n Plots 1132 1110 480 203
Dunholme 556 574 284 120
. Broom's Barn .... . 4610 4522 2610 1668
The results indicate that the amount of alcohol left after Treatments
3 or 4 is slightly inhibitory and that the concentration of alcohol
left in the plates depends on treatment. Because ethanol gave no
better count than glucose or sucrose and was less reliable with our
technique, it was not used in later experiments.
>3
~2
ô
O
~:/ xpt
. . . . , , , ,
0 0.0,4 0.08 0.12 0.15 0,20 0.30 0,40 0.$0 0.60
0.10, 0.12. 0.14, 0.18, and 0.20 per cent. In the second e x p e r i m e n t
the count from clear m e d i u m was significantly higher at low
K 2 H P 0 4 concentrations only (0.02 and 0.04). High concentrations
(0.4 and 0.6 per cent) inhibited Azotobacter. Low availability of
phosphate in the precipitated m e d i u m was p r o b a b l y not the cause
of the low A z o t o b a c t e r count, because this occurred over too wide a
concentration range. The possibilities t h a t the K p a r t of the mol-
ecule or the p H of the m e d i u m at high K 2 H P 0 4 levels were inhibiting
Azotobacter were investigated and found not to affect the count.
DISCUSSION
SUMMARY
less a c c u r a t e b u t m o r e c o n v e n i e n t w h e n m a n y soil s a m p l e s h a v e t o be
examined.
ACKNOWLEDGEMENTS
W e w i s h t o t h a n k Dr. P. S. N u t m a n f o r h i s i n t e r e s t in t h i s w o r k a n d Miss
M. S a n d e r s for h e r t e c h n i c a l a s s i s t a n c e .
REFERENCES