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P l a n t a n d Soil X V l I , no.

3 D e c e m b e r 1962

STUDIES ON AZOTOBACTER SPECIES IN SOIL


1. C O M P A R I S O N O F M E D I A A N D T E C H N I Q U E S FOR
COUNTING AZOTOBACTER IN SOIL

b y MARGARET E. BROWN, SUSAN K. B U R L I N G H A M , and


R. M. JACKSON
Rothamsted Experimental Station, Harpenden, Hertfordshire

INTRODUCTION

Azotobacter species are widely distributed in solls of pH above 6.6


and many authors have described their isolation b y enrichment
teehniques, but few have eonsidered the best methods and media
for counting Azotobacter. J e n s e n 6 found that an agar medium
eontaining dextrin as the carbon source, mineral salts and 5 g/1 of
ehalk, was suitable for counting Azotobacter from soil, but in later
physiological experiments he substituted mannitol for dextrin
( J e n s e n 7 s) and used only 2 g/1 ehalk. Mannitol has been the sub-
strate most favoured for experimental work with these bacteria,
but it may not give the highest count when used in isolation media.
D a r z n i e ' k 4 found that sucrose gave a eount 2 to 3 times higher
than mannitol, and C o l l i n s a got five times the plate count with
Martin's medium eontaining mannitol and suerose as with Ashby's
mannitol medium. In further experiments C o l l i n s ~ increased the
count b y shaking the soil suspension with glass beads.
Azotobacter have also been counted from soll in liquid media.
T c h a n 10 used a simple medium containing mineral salts and 1%
glucose. A u gi e r I favoured a complex medium containing mannitol,
which he claimed prevented gas formation and gare a thicker Azoto-
bacter pellicle than glucose medium. He did not compare the two
media for counting.
We have examined the current methods for counting Azotobac-
ter and paid special attention to techniques and the influence of

- - 3 0 9 - -
B10 M. E. BROWN, S. K. BURLINGHAM, AND R. M. JACKSON

the energy sources in the medium. The most widely distributed


species in soil is Az. chroococcum and most strains isolated by current
methods belong to this species. As far as is known none of the above
media are selective for species and in our work no distinction is
made between them.

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

J e n s en 6 used a medium containing a carbon source, soluble mineral salts


including K2HPO4 and 5 g/1 CaCOa. When using a similar medium we noted
that during autoclaving some of the K2HPO4 precipitated. This medium was
replaced by a clear one containing glueose 5.0 g; 1VigSO4.7H20 0.2 g; FeSO4.
7H20 0.04 g; Na2MoO4 0.005 g; CaC12 (anhydr.) 0.15 g; agar 15.0 g; distilled
water 1000 ml all autoclaved together at 15 lb for 15 min, and 0.8 g/1
K2HPO4 added from a separately sterilised solution to the agar when it had
cooled to 42 °, just before pouring the plates. This modification avoided dis-
tributing precipitated phosphate and CaCOa unevenly in the plates, and
caramelization of glucose, which would have occurred if glucose and phos-
phate were sterilized together. The final pH was 6.8-7.0.

2. D i l u t i o n t u b e m e t h o d . Two-fold dilution series were prepared


from 2-ml aliquots of the initial 10-g soil samples in 100 ml sterile distilled
watet. The series were prepared in a medium similar to that used for the plate
method except agar was omitted and CaCOa (5.0 g/i) replaced the CaC12. A
high concentration of CaCO3 was used to ensure t h a t each tube received a
small quantity from the mechanieal dispenser; variation in the amount
delivered could not be avoided. The tubes were incubated at 25 ° for 14 days
and then read as positive or negative according to turbidity. The tubes at
the end of the series were examined mieroscopically for growth. The num-
bers of Azotobacter per g dry soll were finally calculated by reference to
most probable number tables in F i s h e r and Y a t e s Statistical Tables 5
To conserve pipettes the use of siliconed ones was tested. The inside of
the pipette was coated with a layer of silicone (Repeleote, BDH), which de-
creased the retention of soll suspension droplets on the glass. The dilution
was mixed by rinsing up and down the pipette 4 times and then an aliquot
transferred to the next level of diluent leaving no droplets of suspension
inside the pipette. The same pipette was again used in the same way for the
hext dilution and so on using only one pipette for the complete dilution series.
Comparative tests showed that there were no signifieant differences between
counts made by diluting with this method and by changing pipettes for eaeh
serial dilution.

RESULTS

Experiments on the preparation o/ the soil suspension


S e v e r a l m e t h o d s of p r e p a r i n g t h e soil s u s p e n s i o n were t e s t e d t o
d e t e r m i n e w h e t h e r m o r e A z o t o b a c t e r were c o u n t e d f r o m a h i g h l y
d i s p e r s e d s u s p e n s i o n t h a n f r o m one less v i g o r o u s l y t r e a t e d . T h e
effect of t i m e for w h i c h t h e soil was h e l d i n s u s p e n s i o n b e f o r e
p l a t i n g , t h e t y p e of s u s p e n d i n g f l u i d a n d t h e t i m e of s h a k i n g were
also s t u d i e d . T e n - g r a m s a m p l e s of f r e s h - s i e v e d G a r d e n P l o t soil
were s u s p e n d e d i n e i t h e r t a p w a t e r , d i s t i l l e d w a t e r or phy.siological
312 M. E. B R O W N , S. K. B U R L I N G H A M , A N D R. M. J A C K S O N

saline, shaken for 10 min and plated immediately. Different times


of shaking in distilled water (10, 20, 30 min) followed by immediate
plating were also examined. In a large experiment the suspensions
may stand for as long as 2 h after shaking before plating, during
which time Azotobacter cells could multiply. To test this possibility
suspensions which had stood for 1 and 2 h were plated. The effect of
grinding the soil in a mortar for 2 min before suspending in distilled
water was also examined. None of these treatments had any signifi-
cant effect (P = 0.05) on the count of Azotobacter, which ranged
from 1200 to 1500 per g soil.
In view of C o l l i n s 'a report, mentioned above, four experiments
were made on suspending Garden Plot soil in distilled water with
10-g glass beads (5 to 6 mm diameter) and shaking for different
times (2.5, 5, 10, 20, and 40 min) followed by immediate plating.
These treatments were compared eaeh time with the standard
treatment, 10 min shaking in distilled water followed by immediate
plating, and once with mixing the suspension in a M.S.E. top mace-
rator and removing the samples after 2.5, 7.5, 15, and 30 min bien-
ding. Table 1 gives the results from 4 replicated plates of each
treatment and shows that shaking with glass beads for only 2.5 min
can significantly increase the count. There is no significant increase
when shaking with beads is prolonged beyond 2.5 min. Maceration
does not affect the count.
TABLE 1
Number of Azotobacter per gram dry soll obtained by different soil suspension
treatments
Treatments
E x p t Standard I M.S.E. top macerator ] Glass beads
No. Time of shaking, min p =
10 [2.sl 7.5 I 15 I 3o I 2"5i 5 I 10 ] 20 t 40 0.05
1 1075 835 1085 939 1090 [ - -
i
1948 1767 1810 1810 404
2 1012 920 980 916 880 1080 187
3 656 1339 1267 1421 1189 -- 204
4 752 1246 1264 1321 1351 1338 439

Dispersing agents, Tween 80 (Honeywill-Atlas Ltd.) and Noni-


det P42 (Shell Chemical Company Ltd.), were added to distilled
water in the presence or absence of glass beads, the soil suspension
shaken for 10 min and the treatment compared with the standard.
Table 2 gives results of three experiments and shows that the dis-
METHODS FOR COUNTING AZOTOBACTER 313

persing agents can give contrary results in different experiments.


Nonidet can be toxic and Tween 80 only once significantly increased
the count over that with beads alone (Expt. I). The maximum count
was usually obtained by shaking the soil suspension in distilled
TABLE 2

N u m b e r of A z o t o b a e t e r p e r g r a m d r y soil as affected b y dispersing a g e n t s


Treatments
=
D i s p e r s i n g a g e n t , eonc., tal/100 ml
Standard Beads Beads +
0.01 0.1 0.25 0.5 1.0
0.01 i 0.1 I 02s I 0.5 I 1.0
Nonidet
1. 180 480 -- -- 280 -- 377
2. 524 1216 -- 375 179 89 566 274 338
i
T w e e n 8o Beads +
1. 180 480 408 -- 684* I
2. I 524 1216 -- 417 500 417 1245 506 638
3. 206 729 118 212 613 -- -- 676 613 553

water with beads and the dispersing agents had no further effect;
but unnecessarily complicated the method.

Comparison o/ carbon sources in the medium


Mannitol, glucose, and sucrose were compared as carbon sources
in agar media. They were added from sterile solutions to the bulk
of the sterilized basal medium, to give a final eoncentration of 5.0
g/1 just before pouring the plates. Table 3 gives the counts from four
different soils inoculated on to 6 replieate plates of each medium.
TABLE 3

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

With all four soils significantly more Azotobacter were counted


on glucose and sucrose than on mannitol media. This m a y be be-
cause a proportion of the colonies developing on glucose or sucrose
cannot use mannitol, or because Azotobacter grows quicker on
314 M. E. B R O W N , S. K. B U R L I N G H A M , AND R. M. J A C K S O N

g!ucose a n d sucrose and so decreases possible competitive or anta-


gonistic effects of other micro-organisms. To test the first possibility
50 random colonies from glucose, sucrose and mannitol isolation
plates were inoculated on to further plates of each of the 3 media.
All grew on all media showing that, at least with impure cultures
and mass inocula, all glucose and sucrose isolates can use mannitol.
Glucose was chosen as the best substrate because the Azotobacter
colonies developed quickly and were sharply defined, otherwise with
sucrose the colonies were very "wet" and ran together easily.
N o r r i s (personal communication) obtained high counts of Azo-
tobacter agilis with ethanol and we tested this substrate. Alcohol
is lost b y evaporation while the soll suspension is being dried on
the agar surface. To see whether this affeets Azotobacter counts
ethanol plates were prepared in four ways. Ethanol (0.2 ml) was
added to the plates 1) before first drying the plates, 2) after first
drying but before inoculating, 3) before first drying and with the
inoculum, 4) after inoculating when the plates were complëtely dry,
and the lids were then replaeed immediately. Table 4 compares
counts from these treatments with those from basal medium +
glucose and basal medium without any added carbon source.
TABLE 4
Numbers of Azotobaeter per gram dry soil on ethanol media prepared in
different ways
EthanoI t r e a t m e n t
Glueose No earbon
1 I æ I 3 I '~
3786 I 3906 ] 2540 I "1589 3642

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.

The effect o/drying agar ptates


When agar plätes are surface dried, the concentration of salts
in the medium alters, the diffusion in the liquid phase is decreased
a n d gaseous diffusion is increased. Such factors might affect the
slow-growing colonies on mannitol more than the quick-growing
colonies on glucose.
METHODS FOR COUNTING AZOTOBACTER 315

Glucose and mannitol agar plates were prepared each containing


exactly 10 ml medium. Suspensions from 3 solls were inoculated
on to the surface of the plates which had either not been predried
or had been predried at 25°C for ! or 2 hours. The soll suspensions
were allowed to dry before the plates were closed, all plates of one
drying time being shut together. In spite of the varying length of
the predrying period the total drying time was very similar for all
treatments, usually 2½ to 3 hours.
TABLE 5
Effect of drying time on Azotobacter counts
Glucose Mannitol
Soil Drying time, hours Drying time, hours
0 L { I I 1 2 LP=0.05 0 I ~ i i ] 2 l P=0.05
Allotment 6523 6585] 6586 I 6900 2572 1443 1004 I 1505 821
Garden Plot 2948 3071 2935 2886 1094 482 402 328 396
Broom's Barn 907 1920 1120 1333 1444 800 267 213 373 692

With glucose medium the counts of Azotobacter from any soll


were not infiuenced b y the drying treatment, but with mannitol
predrying lowered the count in all (significantly for allotment and
garden plots). Even without predrying the mannitol medium gave
a rauch lower count than the glucose, which is a further reason for
its rejection as a substrate.

Effect o/phosphate concentration in the medium


J en s e n 8 found that nitrogen fixation b y Azotobacter was great-
ly influenced b y the available phosphate concentration. Counts of
Azotobacter from soil may also be affected by the phosphate con-
centration in the nitrogen-deficient medium. In autoclaved whole
medium some phosphate m a y be precipitated and unavailab]e, the
amounts differing b e t w e e n batches of media and replicate plates.
Possible effects that this might have were examined in two ex-
periments in which two series of media were prepared containing
mineral salts, hut no glucose and to which different amounts of
K2HP04 were added, in one series before autoclaving and in the
other at identical levels just before pouring the ptates; glucose was
also a d d e d at this time.
Figure 1 shows that the Azot0bacter count was little affected b y
total PO4 over a wide range of concentrations (0.02 to 0.6 per cent)
316 M. E. BROWN, S. K. BURLINGHAM, AND R. M. JACKSON

in the precipitated m e d i u m but it was significantly lower t h a n the


count from clear m e d i u m at concentrations of 0.01, 0.05, 0.06, 0.07,

>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

K2HPO4 concentration, ~/o


Fig. I. The effect of K2ttPO4 concentration upon Azotobacter count in
precipitated and clear media.
× - - - × Precipitated medium.
• • Clear medium
@I
Indication of significant differences between counts from the two
x media at any one particular K2HP04 concelltration.

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.

Comparison o/ the agar@late and dilution-tube methods [or counting


Azotobacter
Per replicated unit the dilution-tube m e t h o d is less accurate t h a n
the plate-count method. H o w e v e r when m a n y soil samples have to
be examined at one time it is rauch more convenient, easily read
and more economical of media and incubator space. The two m e t h o d s
METHODS FOR COUNTING AZOTOBACTER 317

were compared; also, because more colonies develop on glucose agar


than on mannitol agar, these substrates were tested in liquid media.
A suspension of allotment soil was plated on glucose and mannitol
agar and two-fold dilution series prepared in glucose and mannitol
liquid media from the same suspension, 6 replicate tubes were inoc-
ulated for each level. Plates and tubes were incubated, the plates
counted after 5 and 7 days and the tubes after 14 days. Table 6 gives
the results of 5 experiments and shows that, regardless of substrate,
the tube count was usually lower than the plate count and that
although the eount on mannitol agar was always significantly
below that on glucose agar, the difference between the two sub-
strates was less and did not always show in liquid media.
TABLE 6
Comparison of Azotobacter counts by the plate-method and dilution-tube method
No. Azotobacter/g dry soil
Agar-plate method Dilution-tube method
Mannitol ] Glucose I Sign. MannitoI I Glucose 1 Sign.
1. 670 3042 1205 1799 n.s.
2. 1817 625 1247 n.5.
3. 1804 3076 1678 6793 S.
4. 1831 4715 728 1239 n.s.
5. 3945 18,033 1126 565 S.

DISCUSSION

Although Azotobacter is widely distributed in non-acid soils, the


reported populations are very small compared to many other soil
micro-organisms. This could be apparent rather than real if the
current counting methods fail to show the full numbers of cells,
either because the technique of preparing soll suspensions is inad-
equate or an unsuitable selective medium is used. We have tested
various techniques and confirmed C o 1lin s' observation that shaking
soll suspensions with glass beads increases the number of colonies
produced. Simple shaking or prolonged treatment of the soff
suspension in a macerator has no effect. S t a r k e y 9 using C h o l o d -
n y's 2 contaet slide method identified cells of Azotobacter, appar-
ently in small aggregates in the soil. The vigorous pounding
action of the glass beads probably breaks these aggregates into
separate cells and adding Tween 80 once helped in their further
dispersal. This suggests that the cells must be firmly attached to
318 M. E. BROWN, S. K. BURLINGHAM, AND R. M. JACKSON

each other, possibly b y their mucilaginous cells walls, and the


problem of separating them is purely mechanical.
Counts of Azotobacter depend greatly on the carbon source and
are smaller with mannitol in agar than with glucose or sucrose.
Drying the plates before Jnoculation decreases the number of colo-
nies on mannitol more than drying afterwards, but this cannot
be the only cause of the poor growth. Dryiflg is an essential part
of the plate method, which means that mannitol should not be used.
The failure of some Azotobacter to develop on mannitol poses the
question of whether there are two distinct populations in the soff;
this is not fully answered b y the reported experiments but such a
possibility is being investigated.
Azotobacter counts are influenced b y the phosphate concen-
tration in the medium. Concentrations above 2 g per litre depress
the count and precipitated phosphate can be deleterious.
Comparison of the agar-plate and dilution-tube methods shows
practical difficulties in both, but the former is more accurate.
Glucose is a suitable substrate for either method; clostridia do not
interfere provided CaCO3 is added to the dilution tubes as a buffer,
but otherwise they produce butyrie acid which inhibits Azotobac-
ter. The results from the dilution-tube method vary more than from
the plate method, particularly when mannitol is used as a substrate.
The techniques and media we describe give more than twice as
many Azotobacter as earlier methods. Even so the numbers ob-
served for soff are still small, but there is no reason to suppose that
they are not valid estimates of the population. The near agreement
between plate and dilution methods, where conditions for microbial
interaction differ greatly, suggests that antag0nism is not imporLant.

SUMMARY

M e t h o d s for c o u n t i n g Azotobacter species in soil h a v e b e e n e x a m i n e d .


T h e h i g h e s t c o u n t s were o b t a i n e d f r o m soil s u s p e n s i o n s shakeri in sterile
distilled w a t e r c o n t a i n i n g 10-g glass b e a d s a n d p l a t e d on to glucose agar.
M a n n i t o l h a s b e e n r e j e c t e d as a s u i t a b l e s u b s t r a t e in a g a r m e d i a b e c a u s e
it gives lower c o u n t s of A z o t o b a c t e r t h a n glucose, all effect w h i c h is f u r t h e r
e n h a n c e d b y d r y i n g t h e a g a r plates. A clear m e d i u m free f r o m p r e c i p i t a t e d
p h o s p h a t e a n d CaCO8 is r e c o m m e n d e d for t h e a g a r - p l a t e m e t h o d ; t h e Azoto-
b a c t e r e o u n t is affected b y t h e p h o s p h a t e c o n c e n t r a t i o n .
T h e a g a r - p l a t e a n d d i l u t i o n - t u b e m e t h o d s »vere c o m p a r e d , t h e l a t t e r is
METHODS FOR C O U N T I N G AZOTOBACTER 3 t9

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 .

Received November 30, 1961.

REFERENCES

1 A u g i e r , J., A propos de la numération des Azotobac~er en milieu liquide. Ann. Inst.


Pasteur 91, 759 765 (1956).
2 C h o l o d n y , N., Über eine neue Methode zur Untersuchung der Bodenmikroflora.
Arch. MikrobioL 1, 620-652 (1930).
3 C o l l i n s , F. M., The occurrence of Azotobacter in two south Australian solls. Austra-
lian J. Exptl. Biol. 30, 587=595 (1952).
4 D a r z n i e k , Yu. 0., The earbon source in a ~medium for the quantitative determina-
tion of Azotobacter in soil. Mikrobiologiya 28, 397-398 (1959).
5 F i s h e r , R. A. and Ya'tes, F., Statistical tables for biological, agrieultural and me:
dical research. 5th ed. Oliver and Boyd (1957).
ó J e n s e n , H. L., Contribntions to the nitrogen economy of AustraIian wheat soils,
with particular reference to New South Wales. Proc. Linn. Soe. New South Wales
60, 1-122 (1940).
7 J e n s e n , H. L., Notes on the biology of Azotob~zcter. Proc. Soc. Applied Baeteriol.
14, 89-94 (1951).
8 J e n s e n , H. L., The nmgnesium requirements of Azotobacter and Beijeri*~cl~ia, with
some additional notes on the latter genus. Acta. Agr. Seand. 4, 224-236 (1954).
9 S t a r k e y , R. L., Some influences of the development of higher plants upon the
miero-organisms in the soil. Microseopic examination of the rhizosphere. Soil Sei.
45, 207-250 (1938).
10 T c h a n , Y. Tl, Studios on nJtrogen Iixing bacteria. 1. A note on the estimation of
AzoEobacter in the soil. Proc. Linn. Soc. New S0uth Wales 77, 89-91 (1952).

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