Isolation of Azotobactor sp. and Azospirillum sp. from the rhizosphere soil and root samples

ISOLATION OF AZOTOBACTOR AND AZOSPIRILLUM FROM SOIL AND ROOT SAMPLES

AIM: To isolate the Azotobactor sp. and Azospirillum sp. from the given rhizosphere soil and root samples.

PRINCIPLE:

                Azospirillum live as symbiotic with plants in the rhizosphere. The plant stimulatory effect exerted by Azospirillum has been attributed to several mechanisms, including biological nitrogen fixation and production of plant growth promoting substances. It has been described to the bacterial production of plant growth regulating substances like plant hormones. An increased number of lateral roots and root hairs enlarge the root surface available for nutrients. This results in higher nutrient uptake by inoculated roots and an improved water status of the plant, which in turn could be the main factor for enhancing plant growth. Azospirillum is not only able to fix atmospheric N but also to mineralize nutrients from the soil, to sequester, Fe, to survive to marsh environmental conditions, and can help plants minimize the negative effects of abiotic stresses.

Azotobacter sp, are free-living aerobic bacteria dominantly found in soils, present in alkaline and neutral soils. They are nonsymbiotic heterotrophic bacteria capable of fixing an average 20kg N/ha/year. Besides, it also produces growth promoting substances and is shown to be antagonistic to pathogens. Azotobacter sp. are found in the soil and rhizosphere of many plants and their population ranges from negligible to 104 g-1 of soil depending upon the physico-chemical and microbiological (microbial interactions) properties. Besides, nitrogen fixation, Azotobacter also produces thiamin, riboflavin, indole acetic acid and gibberellins. When Azotobacter is applied to seeds, seed germination is improved to a considerable extent, so also it controls plant diseases due to above substances produced by Azotobacter. The exact mode of action by which Azotobacteria enhances plant growth is not yet fully understood. Three possible mechanisms have been proposed: N2 fixation; delivering combined nitrogen to the plant; the production of phytohormone-like substances that alter plant growth and morphology, and bacterial nitrate reduction, which increases nitrogen accumulation in inoculated plants. Bacteria isolated from soil rhizosphere by using serial dilution on selective media for Azotobacter (LG medium). Isolates were characterized by morphological & biochemically according to Bergey’s Manual method, to shown properties of Azotobacter spp. 

MATERIALS REQUIRED:

All the media components which has been mentioned in the procedure, glasswares such as test tubes, conical flasks, slides, inoculation loops, Staining kits, Microscopes.

PROCEDURE: Bacterial isolation and identification.

AZOTOBACTOR SP.

1.  Different soil samples from the rhizosphere of agricultural crop were transferred to laboratory.

2.  Strategies used for isolation were: (i) Enrichment of Azotobacter strains, one gram from each of the soil samples were added into 100 ml Erlenmeyer flasks containing 20 ml of Azotobacter broth of the following composition; mannitol 20g, K2HPO4 0.8 g, KH2PO4 0.2 g, MgSO4·7H2O 0.5 g, FeSO4·6H2O 0.10 g, CaCO3 20 g, NaMoO4·2H2O 0.05 g supplemented with ZnSO4.7H2O 10 mg, MnSO4.4H2O 1.0 mg and cycloheximide (100μg/ml) per liter (Adjust to pH 7.2). Incubation was at 28°C for 2-5days.

(ii) Isolation was carried out by preparing serial dilutions from enrichment culture followed by streaking and incubation at 28ºC for 2-5 days. All the isolates were subcultured on selective nitrogen-free specific medium Azotobacter Agar plates.

AZOSPIRILLUM SP.

1.      Soils were collected from various crop fields in and around Coimbatore.

2.      One gram of each collected soil sample was suspended in a tube filled with 5ml of half strength of Winogradsky’s N-free mineral medium containing (g/liter): 10 g of glucose, 25 g of KH₂PO₄, 12.5 g of MgSO₄·7H₂O, 12.5 g of NaCl, 12.5 g of FeSO₄·7H₂O, 0.1 g of Na₂MoO₄·2H₂O, 0.38 g of MnSO₄·H₂O, 0.1 g of CaCO₃, which were sterilized separately, and 15 g of agar, pH 7.2 and then grown at 37⁰C in an incubator shaker overnight.

3.      Serial dilution were made and 0.1 ml aliquots (10^-3 -10^-5) were speeded on plate containing the same agar medium.

4.      The plates were incubated for 2 days at 37⁰C and morphologically different colonies appearing on the medium were isolated.

5.      The isolates were characterized for the following traits: color pigment, form elevation margin, diameter, surface, opacity and texture.

6.      Morphology was evaluated under microscopy and motility was tested by observing the spread of the growth in test tubes of semi agar media.

7.      Biochemical characters of bacterial isolates were examined according to methods described in Bergey’s Manual of Systematic Bacteriology.

RESULTS:(Expected results)

Azospirillum sp. the isolates were microscopically observed for their cell shape and gram reaction. The cell shape of all the isolates was spiral; all the isolates were Gram negative and had cork screw movement when observed under microscope.

Azotobactor sp. After incubation of soil sample in Azotobacter selected media, colony has been thoroughly characterized on the basis of colony color, shape and diameter of the colony. Out of them the characteristics features of some selected colony has been summarized  (Table) Most of the bacterial colony isolated are circular (even) in shape and most of the bacterial colony are whitish in colour and the size ranges between 1.0 – 4.0 mm in size (Table) . Others bacterial colonies isolated are spindle (even) in shape and circular (undulated ) and others bacterial colony are translucent white with central black dot yellowish, and creamy .

isolation and identification of Candida sp.and Aspergillus sp.

Isolation and identification of clinically important fungi Candida albicans & Aspergillus sp.

AIM: To isolate and to identify Candida albicans and Aspergillus sp, from the given specimen

INTRODUCTION: The most common oral fungal infection in human beings is caused by the Candida species. The term Candida originates from the Latin word candid, meaning white. The spores of Candida are a commensal, harmless form of a dimorphic fungus. Its prevalence in healthy human oral cavities. However, when appropriate conditions such as local or systemic deficiencies in the host defenses supervene they become invasive and pathogenic pseudohyphae. Mycotic infections have become a major cause of morbidity and mortality in clinically debilitated or immunocompromised patients. The co-existence of Candida species are humans either as commensals or pathogens. The genus Candida includes several species C. albicans is by far the most common species causing infections in humans. The emergence of non-albicans Candida species as significant pathogens has however been well recognized. Although they are closely related they differ from each other with respect to epidemiology, virulence characteristics, and antifungal susceptibility.

Aspergillus species are filamentous fungi that are commonly found in soil, decaying vegetation, and seeds and grains, where they thrive as saprophytes. Aspergillus species can be occasionally harmful to humans. Most Aspergillus species are found in a wide variety of environments and substrates on the Earth throughout the year. Only a few well-known species are considered as important opportunistic pathogens in humans

MATERIAL REQUIREMENTS:

Sabouraud’s dextrose agar medium, Pre sterilized cotton swabs, Potassium hydroxide, Gram stain kit, Corn meal agar and required glass wares.

PROCEDURE:

Methods of sample collection & Isolation: Candida albicans

1. Smear technique: Scraping and smearing directly on the slide

 2. Plain swab: Using cotton swab sample is collected from the lesional tissue

 3. Impression culture technique: Impression casting in agar fortified with broth. 

4. Concentrated oral rinse: 10 ml of sterile phosphate buffered saline rinsed for 1minute. The solution is then concentrated (10-fold) by centrifugation and 50 ml, inoculated on an agar medium. 

Aspergillus sp.

1.      PDA (peptone dextrose agar) was used as the culture medium while collecting the samples.

2.      30 mg/L streptomycin had been added to the culture to prevent bacteria reproduction.

3.      Rose-bengal stain was added to the culture in order to prevent faster reproduction of moulds

4.      Peptone Dextrose Agar which was used for isolation was put into 7 days of incubation in laboratories at room temperature (22-26 ºC).

5.      After the incubation, pure cultures of microfunguses were obtained. Lactophenol solution stained by picric acid and lactophenol solution stained by cotton blue were used for investigation of microscopic structures of moulds.

Identification of Candida sp.:

 1. Direct microscopy Morphological features of Candida sp. need to be examined for identification.

2. Potassium hydroxide (KOH) preparation of the specimen reveals non-pigmented septate hyphae with characteristic dichotomous branching.

3. A smear taken from the lesional site is fixed on to microscope slides and then stained either by the gram stain or by the periodic acid Schiff (PAS) technique.

4. Laboratory culture of Swab: The sampling approach involves gently rubbing a sterile cotton swab over the lesional tissue and then subsequently inoculating a primary isolation medium such as Sabouraud’s dextrose agar (SDA)

5. Culture media: The most frequently used primary isolation medium for Candida is SDA which, although permitting growth of Candida, SDA is incubated aerobically at 37°C for 24–48 hrs.

6. Morphological criteria: The germ-tube test is the standard laboratory method for identifying C. albicans. The test involves the induction of hyphal outgrowths (germ-tubes) when subcultured in serum at 37 °C for 2-4 hours.

7. Chlamydospores are refractile, spherical structures generated at the termini of hyphae following culture of isolates on a nutritionally poor medium such as cornmeal agar. Agars are incubated for 24-48 hours at 37°C and then examined microscopically for chlamydospore presence

8. Biochemical identification: Candida species is largely based on carbohydrate utilization. Traditional testing would have involved culture of test isolates on a basal agar lacking a carbon source. Carbohydrate solutions would then be placed within wells of the seeded agar or upon filter paper discs located on the agar surface. Growth in the vicinity of the carbon source would indicate utilization.

RESULTS: Morphological characteristics of Candida species.

S.no	Morphological characteristics	Features
1.	Size (μm	3–6.2
2.	Shape	Spherical or oval
3.	Number of buds	Single; chains
4.	Attachment of buds	Narrow
5.	Thickness	Thin
6.	Pseudohyphae &/or hyphae	Characteristic
7.	Number of nuclei	Single

Candida develops as cream, smooth, pasty convex colonies on SDA and differentiation between species is rarely possible.