Isolation and Identification of microorganisms from soil sample

Microbial ecology Lab report

1. Introduction

Life on this planet could not be exist if microbes are absent on earth that have enormous presence in  soil. Microorganisms are essential for degradation of organic matter that is deposited in the soil, such as dead materials of plant and animal tissues and also animal wastes. Breaking down of these organic particles by microbial enzymes provides and replenishes the soil with basic essential nutrients known as growth factor for living bodies. By the process of enzymatic transformations, plants assimilate these elemental nutrients into organic compounds which is require for growth and reproduction. Following this method, plants serve as an essential source of nutrition for animals and human being. So, a plenty of soil borne microbes play a significant role in a number of elemental cycles, such as the nitrogen cycle, sulfur cycle, hydrogen cycle and the carbon cycle. Cappuccino G .James (10th edition).

Microbial populations that found in soil play a significant role in soil properties and effect on  below-ground ecosystem processes. Microbial composition and functioning changes the soil quality through decomposition of organic matter, recycling of nutrients, and biological control of parasites of plants. Moreover, the discovery that soil microbes may translate into benefits for biotechnology, management of agricultural, forest, and natural ecosystems, biodegradation of pollutants, and waste treatment systems maximized the need of scientists for the isolation and their characterization. Stefanis C et al, (2013).

Soil contains numerous species of microorganisms, including bacteria, fungi, protozoa, algae, and viruses. The most existing species are bacteria, including the mold like actinomycetes, and fungi. It is essential to bear in mind that the soil environment differs from one location to another and from one period of time to another. Therefore, factors such as moisture, pH, temperature, gaseous oxygen content, and organic and inorganic composition of soil are crucial in determining the specific microbial flora of a particular sample. Just as the soil differs, microbiological methods used to analyze soil also vary. Cappuccino G .James (10^th edition).

A single technique cannot be used to count all the different types of microorganisms present in a given soil sample because no one laboratory cultivation procedure can provide all the physical and nutritional requirements necessary for the growth of a greatly diverse microbial population. The aim of the present laboratory work was to isolate diverse microbial communities from soil sample. The method used was the serial dilution–agar plate procedure. Nutrient agar media was employed to support the growth of different types of microorganisms such as actinomycetes fungi, and bacteria. Eventually, only the fungal communities were observed from the supplied soil sample under light microscope.

2. Materials and methods

The complete research was conducted into the In-vitro Research Laboratory of University of Oulu, Finland. Supplied soil samples were collected from peat land area by PhD research group and a small portion was used to perform this experiment taken from the storage chamber. The following precautions were carefully taken during collection of the samples: - The samples were collected in the sterile plastic containers. The samples were transported to the laboratory as promptly as possible after collection. Standard microbiological practices were maintained during the sampling time. Samples collected from the particular sites were tested to determine their pH values.

Prior to the inoculation of sample, serial dilution was carried out. At first, 10 mg of supplied soil sample was added to 90 ml of sterile normal saline (0.88% Nacl). It was considered as the first diluted sample. Then 1 ml sample was transferred to 9 ml of normal saline to make the 10-2 dilution. This particular step was repeated till it is reached to 10-6 dilution. By using sterile tips, 0.1 ml from the 10-2, 10-3, 10-4, 10-5 and 10-6 dilutions of each sample was transferred on the Nutrient Agar (NA) plates and spread plate method was employed. All the plates were incubated at 25°C for 24 hours.

Nutrient Agar medium, pH adjusted to 7.3, was used for the isolation of diverse microbes like bacteria, actinomycetes, fungi etc. Nutrient agar medium is also used for cultural characterization of the isolated organisms. After cooling (45°C), approximately 20 ml of culture medium was poured into sterilized Petri plates inside a laminar flow cabinet and allowed to solidify. NA medium was used for cell mass production and prepared by the addition of Yeast Extract.

The standard growth was used in all experiments was Nutrient agar. Petri-plates were incubated by spreading of the inoculums and transferred to incubator for different condition like temperature, pH and time. All the microbes were examined, isolated and characterized depending on their size, shape, color and reproducing system and other morphological characteristics under the light microscope.

3. RESULTS

3.1. Characteristics of the isolates

The morphological and cultural characteristics of different strains isolates from supplied peat soil vary each other. These characteristics have great effect on the isolation of different group of microbes separately. A comparative preliminary investigation on some of these important characteristics of those isolates was carried out.

3.2. Colony Morphology of Microbes

The microbes were grown on nutrient agar plates at 25°C for 24 hours. After that, nutrient agar plates were observed and the microbes showed the following colonial characteristics based on their dilution factors:

Figure 1. Growth of microorganisms on nutrient agar plates after 24 hours of incubation at 25° C.

3.3. Cultural characteristics

The microbes were grown on nutrient agar plates at 25°C for 24 hours of studying their colonial characteristics (size, shape, color, margins, elevation, optical characteristics, etc. A comparative result on the basis of the characteristics of the microorganisms is shown in Table 1.

Table 1. Colonial morphology of the microbes isolated from peat soil sample.

Dilution No.	CFU	Size	Color	Shape	Appeara-nce	Elevation	Margins	Opacity
10-2	TNTC	Large/small	Yellow/ Colorless	circular	Gummy	Raised	Entire	Opaque
10-3	67	Medium/small	Yellow/ Colorless	circular	Gummy	Raised	Entire	Opaque
10-4	8	Large/medium	Yellow/ Whitish	irregular	Cottony	Convex	Filament-ous	Opaque
10-5	4	Large/pinpoint	Yellow/ Whitish	irregular	Cottony	Convex	Undulate	Opaque
10-6	1	Pinpoint	Colorless	circular	Gummy	Raised	Entire	Transp-arent

3.4. Microscopic observation

All microbes from nutrient agar plates were examined under light microscope to evaluate their microscopic appearance before performing gram staining. Interestingly, all colonies from different plates were easily observed under light microscope by using resolution10X, 40X and also 100X. This indicated that no further Gram staining steps is required for this sample. Because all the colonies that grows on nutrient agar plates were fungi. The reason is that without Gram staining bacteria and other microbes are not possible to see under light microscope without staining procedure. So the growths of the microbial colonies on nutrient agar plates were all fungal colonies.

4. Discussions

Several strains of fungi were isolated from soil samples, including both filamentous fungi and yeast from peat land soil samples. Surprisingly, bacterial diversity was absent at that supplied sample. Peter H. Janssen (2002) claimed that plate count methodologies are not suitable for cultivation of soil bacteria and that the members of groups without cultivated representatives are somehow non-culturable. They reasoned that by increasing the culturable fraction of soil bacteria above that generally reported, They should be able to isolate some members of groups that to date have not been grown in pure culture.

Fungi are very abundant in the soil and may represent up to 80% of soil microbial biomass (Kirk et al., 2004). The present experiment also showed the fungal growth in peat land soil sample which reaches 100% of soil microbial biomass. Arbuscular mycorrhizal fungi (AMF) are plant‐root symbionts, and are the most abundant and widely distributed fungi in the soil. Their extensive mycelial network can act as a root extension, which can explore a much larger soil volume than roots themselves, and can therefore increase the plant uptake of essential minerals and water. These fungi also play an important role in plant growth and protection against soil‐borne pathogens (St‐Arnaud & Vujanovic, 2007).

The use of serial dilution liquid culture techniques has proved to be effective for isolating numerically significant members of the community in anoxic rice paddy soil. Chin, K.-J (1999). However, when these techniques were used to cultivate bacteria and fungi from the peat land soil to perform in our laboratory, it showed good growth of microorganisms. But when it was observed under microscope all were showed fungal species and for bacteria it showed null results.

The reason of the null growth of bacterial species might be the effect of storage conditions of soil that we collected from university research laboratory. The supplied sample was taken from storage chamber of University of Oulu laboratory, which was kept there for a long time. And several studies showed that storage has a great effect on the growth of bacteria and also minor effect on fungi. Akpan Godwin (2015) showed that Gram negative bacteria were unable to survive up to the end of the experiment if it is keep into the storage condition for more than 7 days after collection. His research showed that all the enzymes are significantly affected by length of soil sample storage. He said to achieve optimum result in biogeochemical study; soil samples should not be stored more than seven days.

Our experiments resulted in isolation of soil microbes were belonging to both filamentous fungi and yeast, suggesting that we need more laboratory methods to extend the range of culturability among soil bacteria and other spp. We obtained pure-culture isolates of different colors and various appearances, which should now need further upgraded physiological and genetic characterization of representatives of these soil microbial groups and their actual identification to help, elucidate their roles in the soil.

5. References

1. Stefanis C et al, (2013). Principal methods for isolation and identification of soil microbial communities. Folia Microbiol. 58: 61. https://doi.org/10.1007/s12223-012-0179-5.

2. Cappuccino G .James, Sherman Natalie, Microbiology A laboratory manual, tenth edition, Pearson Education.

3. Kirk JL, Beaudette LA, Hart M, Moutoglis P, Klironomos JN, Lee H & Trevors JT (2004) Methods of studying soil microbial diversity. J Microbiol Meth 58: 169–188.

4. St‐Arnaud M & Vujanovic V (2007). Effects of the arbuscular mycorrhizal symbiosis on plant diseases and pests. Mycorrhizae in Crop Production (Hamel C & Plenchette C, eds.), pp. 67–122.

5. Chin, K.-J., D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen. 1999. Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms. Appl. Environ. Microbiol. 65:5042-5049.

6. Akpan Godwin U and Mohammed Iliyasu (2015). Effect of length of soil sample storage on soil microbiological biochemical properties in Uyo, Nigeria. Global Journal of Agricultural Research. pp.25-37.

Written by:

Md. Rayhan Mahmud

Future student, Department of Ecology and Population Genetics

University of Oulu, Finland

M.Sc. in Microbiology, Jagannath University, Bangladesh