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vol.29 número3MICROBIAL ACTIVITY OF DARK RED LATOSOL SAMPLES STORED AT DIFFERENT TEMPERATURES índice de autoresíndice de assuntospesquisa de artigos
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Revista de Microbiologia

versão impressa ISSN 0001-3714

Rev. Microbiol. v. 29 n. 3 São Paulo Set. 1998 



Francisco Cleber Sousa Vieira; Ely Nahas *
Departamento de Microbiologia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, SP, Brasil

Submitted: October 19, 1997; Returned to authors for corrections: February 06, 1998;
Approved: July 23, 1998




The number of colony forming units (CFU) of different groups of bacteria and fungi in samples stored at temperatures of 5 to -12oC for 0-32 weeks was evaluated. The number of CFU obtained after the different periods of storage of red latossol soil was compared with the number of colonies obtained immediately after removal of soil samples (time zero). The number of total bacteria and actinomycetes in the samples remained practically unchanged throughout the storage period. The number of Gram-negative bacteria decreased by as much as 69% compared to control, while the number of Bacillus spp and of fungi increased 1.9 to 4.9 times starting from the 12th week in samples stored at 5oC. Except for the variations observed in fungal counts, the remaining groups of bacteria practically showed the same variation in number of colonies in soil samples stored at 5oC and -12oC.

Key words: bacteria, fungi, phosphatase, soil storage




The degradation of organic matter in soil largely results from the interaction between macro- and microorganisms, although microorganisms have a greater participation in this process because of their higher biomass values (7). However, to determine the microbiological characteristics of soil, samples should be promptly analyzed after collection to avoid the risk of obtaining altered results due to the variation in number of microorganisms when the samples are maintained at room temperature. If microbial analysis cannot be performed immediately after collection, Clark (3) recommends conservation at 4oC for a maximum period of 1-2 weeks. Higashida and Takao (8) performed microbial counts in soil samples up to approximately 8 hours after collection.

In support of this view, Harding and Ross (6) reported that fresh soil samples are desirable to characterize microbial counts. If the procedures can not be carried out immediately, some form of storage should be used. Dunn et al. (4) and Lahdesmaski and Diispanen (10) stored soil samples in sterilized plastic bags at -20oC until the time for processing. Lower temperatures, such as -80oC (5), or higher temperatures, such as 5oC (18) or 25oC (20) have been used in some studies.

Different procedures may be used to evaluate the number of microorganisms, to determine microbial mass or to measure microbial activity (13). Microbial counts on plates by the serial dilution method require a considerable number of steps to obtain the desired result, a fact that renders this method more cumbersome than chemical determination. When the number of samples is large, practical limitations prevent sample processing at one time, which would be the ideal procedure. Thus, there is no other alternative than the storage of these samples and the determination of parameters concerning the microorganisms within possible limits. Our objective was to determine the effects of soil sample storage at temperatures of -12oC and 5oC on the counts of defined groups of microorganisms. The present paper compliments a previous publication that evaluated the effects of these systems on the microbial activities (19).



Soil: the characteristics of the soil and the treatment of soil samples were presented in a precedent paper (19).

Microbial counts: A serial decimal dilution was performed by adding 10 g soil, wet weight, to 95 ml of a 0.1% (w/v) sodium pyrophosphate solution. Aliquots of these suspensions were later transferred to Petri dishes containing specific media for counts of microbial groups. Total bacteria were counted using the medium of Bunt and Rovira (2). The same medium was used for the counts of Bacillus spp spores after inoculation with diluted suspensions heated to 80-85oC in a water bath for 10 min. The same medium supplemented with 5 µg crystal violet was used for counts of Gram-negative bacteria (8). For actinomycete counts we used starch-casein agar medium (9) supplemented with 50 µg/ml nystatin, 50 µg/ml cycloheximide, 5 µg/ml polymyxin-b-sulfate and 1 µg/ml sodium penicillin (21). Fungi were counted in Martin (11) medium supplemented with a mixture of penicillin and streptomycin (0.1 g/l, w/v) and 70 µg/ml rose bengal. Microbial counts were determined by the pour plate method after incubation of the cultures at 28oC for 4 days (total bacteria, Bacillus spp. and Microbial numbers were determined.

Statistical analysis: The data are presented as the means of four samples. Analysis of variance of the completely randomized design were calculated using the STATGRAPHICS PLUS 6.0 software. The number of CFU (colony forming units) was transformed to ln (x+1). All means were compared to control values by the Tukey test, with the level of significance set at 5%.



Total bacterial counts ranged from 1.70 to 2.45 x 107 and from 1.50 to 2.67 x 107/g dry soil for samples stored at 5oC and -12oC, respectively (Table 1). These results show that counts did not differ significantly between these two temperatures. The control count (time zero) was 1.59 x 107/g dry soil. No significant differences were detected in the values of stored samples and samples analyzed at time zero, except for the period of 32 weeks.


Table 1. Influence of temperature and period of storage on total bacteria count.

0001i01.gif (13935 bytes)

** = significant (P<0.01); Values followed by same letter are not significantly different as P < 0.05 (LSD)


The number of Bacillus spp was 0.57 x 106 CFU/g dry soil in the control and increased 1.9 to 4.9 times in the stored samples (Table 2). Colony number ranged from 1.80 to 2.79 x 106/g dry soil in samples stored at 5oC and from 1.06 to 2.74 x 106/g dry soil in samples stored at -12oC, showing that the difference between these storage temperatures was small. These results indicate greater growth and resistance of Bacillus spp to adverse conditions.


Table 2. Effect of temperature and period of storage on Bacillus spp.

0001i02.gif (14400 bytes)

** = significant (P<0.01); Values followed by same letter are not significantly different as P < 0.05 (LSD)


In contrast to what was observed with Bacillus spp, the number of Gram-negative bacteria was decreased by as much as 69% compared to control, except for the periods of 2 (5oC) and 16 weeks (-12oC), with the samples stored at -12oC being the most affected ones (Table 3). The total number of CFU was 8.91 x 105/g dry soil at time zero and ranged from 3.34 to 9.86 x 105/g dry soil in the samples stored at 5oC and from 2.79 to 9.78 x 105/g dry soil in the samples stored at -12oC.


Table 3. Variations in the counts of Gram-negative bacteria after different temperatures and periods of storage.

0001i03.gif (14748 bytes)

** = significant (P<0.01); Values followed by same letter are not significantly different as P < 0.05 (LSD)


As observed for total bacteria, the number of actinomycete colonies did not differ significantly between stored and control samples, except for the 8th week when the counts were reduced (Table 4). While the number of CFU was 3.05 x 106/g dry soil at time zero, a variation from 1.69 to 3.22 x 106 CFU/g dry soil was observed in the samples stored at 5oC and a variation from 1.51 to 3.68 x 106 CFU/g dry soil was observed in the samples stored at -12oC. These data indicate that there was no difference in colony number between samples stored in a refrigerator or in a freezer.


Table 4. Influence of temperature and period of storage on actinomycetes.

0001i04.gif (13574 bytes)

** = significant (P<0.01); Values followed by same letter are not significantly different as P < 0.05 (LSD)


The fungal colony counts showed a different behavior, i.e., while samples stored at -12oC did not differ from the control, those stored at 5oC increased starting from the 12th week (Table 5), indicating that the temperature of 5oC permitted a 35 to 66% increase in number of viable colonies. The control showed 1.16 x 105 CFU/g dry soil, the samples stored at 5oC showed 0.96 to 1.93 x 105 CFU/g dry soil and those stored at -12oC showed 0.70 to 1.72 x 105 CFU/g dry soil.


Table 5. Counts of fungi as influenced by temperature and period of storage.

0001i05.gif (13877 bytes)

** = significant (P<0.01); Values followed by same letter are not significantly different as P < 0.05 (LSD)



In addition to the implications of the determination of number of microorganisms during soil sample storage, one should consider the qualitative aspects of the preservation of important species and groups of microorganisms and of the changes in these biochemical characteristics resulting from the variations in these counts.

The results obtained for total bacteria confirm those reported by Ramsay and Bawden (15) who showed little variation in counts over a period of storage of 98 days at 5oC, except on the 22nd and 36th days. Soulides and Allison (16) reported that soils frozen at -22oC for 24 h presented a reduction in bacterial population. However, these investigators commented that naturally or deliberately frozen soils can withstand low temperatures.

Approximately 60 to 100% of bacterial cells occur in soil as spores and may be more tolerant to extreme temperatures of -5 and 75oC (1). According to Paul and Clark (12), growth may be paralyzed at 0oC depending on the size of the bacterial population. In the present study, the number of Bacillus spp detected was quite low (11% of the mean of the total bacterial population), indicating that conditions were not favorable to the growth of spore-forming bacterial populations. On the contrary, the occurrence of fungal growth after a lag phase at 5oC may be explained by the existence of sporulated forms.

Gram-negative bacteria include many species involved in the processes of organic matter degradation, mineral compound transformation and atmospheric nitrogen fixation in soil (17). The reduction of up to 69% in Gram-negative bacterial counts observed in stored samples indicates probable loss of important species of Gram-negative bacteria, impairing subsequent studies. Harding and Ross (6) observed a decrease in nitrifying bacteria in soils frozen at -20oC for more than 6 months, although there was no loss in mineralizing activity even after 9 months. Higashida and Takao (8) reported that Gram-negative bacteria are more susceptible to environmental factors than other groups of microorganisms. In turn, Perez et al. (14) reported that most Gram-negative and coryneform bacteria developed well at 2oC in vitro, suggesting that these microorganisms may be more important in winter, a result differing from that reported here. The type of soil used may have affected the results obtained.



This work was supported by a grant nº 92/4635-2 from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (EN) and from FAPESP (FCSV).




Contagem microbiana de amostras de latossolo vermelho-escuro armazenadas em diferentes temperaturas

Amostras de solo de um latossolo roxo foram armazenadas à temperatura de 5oC e -12oC, pelo período de 0-32 semanas, com o propósito de se avaliar a influência desses sistemas de conservação nas contagens de diferentes grupos de bactérias e fungos. O número de unidades formadoras de colônias (UFC) nos diferentes períodos de armazenagem foi comparado com o número de colônias obtido logo após a retirada das amostras do solo (tempo zero). O número de bactérias totais e de actinomicetos permaneceu praticamente invariável durante o período de armazenagem das amostras de solo. O número de bactérias Gram-negativas diminuiu em relação ao controle em até 69% enquanto o de Bacillus spp aumentou de 1,9 a 4,9 vezes, assim como o de fungos, a partir da 12a semana nas amostras armazenadas à temperatura de 5oC. Excetuando as variações observadas nas contagens de fungos, os demais grupos de bactérias mostraram, praticamente, a mesma tendência de variação no número de colônias nas amostras de solo armazenadas à temperatura de 5oC e -12oC.

Palavras-chave: bactérias, fungos, fosfatase, armazenagem de solo.




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* Corresponding author. Mailing address: Departamento de Microbiologia, FCAV/UNESP. Rod. Carlos Tonanni, Km 5, CEP 14870-000 Jaboticabal, SP, Brasil; Fax: (+5516)322-4275; E-mail:

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