Vol. 44, No. 5
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1982, p. 1246-1247
Copyright C 1982, American Society for Microbiology
Comparison of the Pour, Spread, and Drop Plate Methods for
Enumeration of Rhizobium spp. in Inoculants Made from
H. J. HOBEN AND P. SOMASEGARAN*
NifTAL Project, Department ofAgronomy and Soil Science, College of Tropical Agriculture and Human
Resources, University ofHawaii, Honolulu, Hawaii 96822
Received 9 March 1982/Accepted 15 July 1982
Inoculants prepared with presterilized peat were enumerated by the pour,
spread, and drop plate techniques. Results indicated that the three plating
methods were interchangeable. The drop plate method was preferred because of
its economy in materials and labor.
In the quality control of peat inoculants, the
numbers of viable rhizobia are determined by
plating on agar medium or by the plant infection
method (3). With inoculants prepared from pre-
sterilized peat, viable numbers of rhizobia may
be determined by the pour, spread, and drop
plate methods. When large numbers of samples
are plated, as happens during routine quality
control checks, the method chosen should econ-
omize on the plating media and petri dishes. The
method should also allow easy execution of the
plating process without sacrificing the accuracy
of the results.
The pour and spread plate methods are exten-
sively used for viable counts with pure cultures
ofRhizobium spp. The drop plate method (2, 3),
however, is less frequently used, and no infor-
mation exists on this method with regard to its
relative recovery efficiency in comparison to the
pour and spread plate methods.
To compare these plating methods, we recov-
ered rhizobia from 10 different peat inoculants.
Each strain ofRhizobium was grown individual-
ly in 100 ml of yeast mannitol broth (3) in 250-ml
Erlenmeyer flasks. Flasks were shaken on a
rotary shaker (100 rpm); the incubation tempera-
ture was 28°C.
Finely ground (mesh size, 200) peat was pur-
chased from the Nitragin Co., Milwaukee, Wis.,
and adjusted to pH 6.5 to 6.8 with precipitated
calcium carbonate. The peat was then packaged
in 50-g quantities in polyethylene bags (16.5 by
11.5 by 0.0038 cm) and sterilized by gamma
irradiation (2.5 megarads). A 50-mi sterile plastic
syringe (Becton, Dickinson & Co., Rutherford,
N.J.) fitted with an 18G 1.5-in. (3.8-cm) needle
t Journal Series article no. 2700 of the Hawaii Institute of
Tropical Agriculture and Human Resources.
was used to introduce aseptically 40 ml of a
turbid culture (ca. 109 cells per ml) into the peat.
The needle puncture on each bag was sealed
with adhesive tape. The bags were thoroughly
massaged to incorporate the culture into the peat
and incubated at 28°C for 1 week. At sampling, 1
g of the inoculant was removed aseptically and
transferred into 99 ml of quarter-strength yeast
mannitol broth in milk dilution bottles. The
bottles were shaken (wrist action shaker) for 15
min. For each strain, five dilution series were
prepared from the same original suspension.
From each series, portions of 0.03 ml, 0.1 ml,
and 1.0 ml of the appropriate dilutions were
plated (in duplicate) for the drop, spread, and
pour methods, respectively. The plating medium
was yeast mannitol agar (3) containing Congo
red (0.25% [wt/vol]). For the drop method, each
plate was divided into eight sectors, and 1 drop
(0.03 ml per drop) was delivered to each sector
with a calibrated pasteur pipette. The plates
were dried at 28°C for at least 2 days before
plating by the spread and drop methods. The
agar was cooled to 50°C (3) and dispensed at the
rate of 15 to 20 ml per plate for pour plating.
The results of viable counts of the inoculants
by the three methods are shown in Table 1.
There was no general trend in the data; no one
method consistently gave the highest or the
lowest counts. This suggested that any one of
the three methods may be adopted for routine
With pour plates, agar cooled to 45°C may
result in selective killing of heat-sensitive strains
of bacteria and therefore lower the counts with
the pour plates (1). In this investigation, al-
though the agar was cooled to 50°C, the pour
plating did not consistently result in the lowest
counts, except in four instances.
TABLE 1. Comparison of drop, spread, and pour plate techniques for the enumeration of rhizobia in peat
LOg1o no.ofrnlzooia per gramofmoist peat,
Mean of five replications. Means in boldface type within each column are significantly different (P
according to the Duncan multiple range test. Laboratory sources were as follows: TAL, NifTAL Project,
University of Hawaii, Honolulu; ICRISAT, International Crop Research Institute for the Semi-Arid Tropics,
Hyderabad, India; NIT, Nitragin Co., Milwaukee, Wis.; CB, Commonwealth Scientific and Industrial Research
Organization, Brisbane, Australia; and USDA, U.S. Department of Agriculture, Beltsville, MD. The rhizobia
were isolated from nodules of the following legumes: TAL22, Phaseolus lunatus; TAL82, Leucaena leucoce-
phala; TAL437, Vigna radiata; ICRISAT3889, Cicer arietinum; TAL1000, Arachis hypogaea; TAL182,
Phaseolus vulgaris; NIT176A22, Vigna unguiculata; CB756, Macrotyloma africanum; USDA136b, Glycine
max; and TAL999, Vigna unguiculata.
Of the three methods, the drop plate proce-
dure was the most preferable because more
counts could be made on one plate (Fig. 1). This
FIG. 1. Growth of colonies of Rhizobium
(TAL437) from drops plated by the drop method. Two
dilutions (10-6 and 10-') of the peat inoculant were
plated in quadruplicate and incubated at 28°C for 7
days. Note the reproducibility in all four drops of the
was because the division of one plate into eight
sectors was equivalent to producing eight spread
or eight pour plates. Furthermore, with dupli-
cate plating ofeach dilution, four dilutions could
be plated on each plate. There was considerable
economy in the use ofavailable incubator space,
and the drop method was also less laborious to
carry out than the other two methods. These
advantages were significant enough to cause us
to adopt the drop plate method for quality con-
trol of inoculants prepared from presterilized
peat. In situations in which inoculants have been
exposed to high temperatures, the plant infec-
tion method may be preferred over plating meth-
ods for a reliable estimate of the numbers of
infective rhizobia (4).
This work was supported by U.S. Agency for Internal
Development contract ta-C-1207 (NirTAL Project, University
1. Clark, D. S. 1967. Comparison of pour and surface plate
methods for determination of bacterial counts. Can. J.
2. Miles, A. A., and S. S. Misra. 1938. The estimation of the
bactericidal power of blood. J. Hyg. 38:732-749.
3. Vincent, J. M. 1970. A manual for the practical study of
root-nodule bacteria. IBP (Int. Biol. Programme) 15:54-58.
4. Wilson, D. O., and K. M. Trang. 1980. Effects of storage
temperature and enumeration method on Rhizobium spp.
numbers in peat inoculants. Trop. Agric. (Trinidad) 57:233-
VOL. 44, 1982