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.

AMYLASE PRODUCTION AND ASSAY

AMYLASE PRODUCTION AND ENZYME ASSAY

AIM: To isolate amylase producing organism from the soil sample and assay their production.

PRINCIPLE: Amylase is the enzyme which breaks down starch into glucose molecules and commonly called as glycoside hydrolase enzymes. Amylase is an enzyme that is used in various industries to rapidly degrade complex polysaccharides (e.g. starches) into smaller oligosaccharides. Starch is an abundant carbon source in nature, and -amylase (1, 4-a-D- glucanohydrolase), which hydrolyzes a-1, 4-glucosidic linkage in starch-related molecules, is one of several enzymes involved in starch degradation. Amylases are among the most important industrial enzymes and also have great significance in Microbiology studies. We screened soil bacteria to produce an amylase in media. Bacterial and fungi strains isolated from garden soil were tested for its abilities to hydrolyze the structural polysaccharides. The strain grows well at 37^o C and the 2% starch concentration, with PH near neutral. The enzyme activities were observed at 2% starch concentration. Amylase activity was assayed by measuring the amount of reducing sugars released from starch using dinitro salicylic acid method.

MATERIALS REQUIRED: Starch agar, Iodine Solution, Dinitro salicylic acid, Maltose, Sodium Phosphate Buffer, Starch solution

PROCEDURE:

1.      Isolation and primary screening for amylase producers was done by using starch agar (containing 1% starch and 2% agar) plate method.

2.      Sediment samples were serially diluted up to 10^-4 and 0.1 ml the diluted samples were spread over the surface of starch agar medium.

3.      Plates were incubated at 30^oC for 24 hrs. Morphologically different colonies were selected for the secondary screening.

4.       In screening, 50 µl of cell free culture was inoculated in the wells made in starch agar medium. The plates were incubated at 30^oC for 48 hrs.

5.      After incubation, the plates were flooded with 1% of iodine solution for 5 min and washed with water to remove the excess color.

6.      Based on the highest size of zone of clearance around the well the potential strain was selected and maintained on starch agar slant.

7.      Enzyme assay by DNS method: The crude enzyme obtained after centrifugation was assayed for amylase activity by measuring the release of reducing sugar following the DNS method.

8.      Preparation of Maltose standard curve: A stock solution of 1mg/ml maltose was prepared in 0.1M sodium phosphate buffer (pH 7.0) and diluted. The graph was plotted between different concentration of maltose and their respective O.Ds

9.      Enzymatic assay of amylase: One ml of crude enzyme supernatant was taken in test tube and 1.0 ml of substrate (starch solution) was added in test tube. The test tubes were covered and incubate at 35°C for 15 minutes in water broth. Then 2.0 ml of DNS reagent was added in each tube and the reaction was stopped by boiling the reaction mixture in water bath for 10 minutes. After cooling at room temperature, the absorbance (O.D) was measured at 540 nm by spectrophotometer and the released sugar was determined from maltose standard curve. One unit of amylase activity was defined as the amount of enzyme that released 1µmol reducing sugar equivalent maltose per minute under the assay condition. The amount of enzyme produced was expressed as μgm / ml.

10.  Calculation: Calculate the amount of reducing sugar present in the sample using standard graph.

11.  Estimation of glucose: The amount of glucose present per ml in amylase assay mixture was calculated from the standard graph.

RESULTS AND DISCUSSION:

Morphologically different strains were selected for amylase production screening. Based on the screening, No. ------------------- Isolates were selected for the amylase production.

Table 1:-No. of strains and their characteristics

The screening using well assay to isolate efficient strain and performed enzyme assay per ml of production by DNS method.

Table 2: The OD values were tabulated based on their respective dilution.

Photos:

Give the appropriate title to the figures and tables

                                                  

protease production and assay

PROTEASE ENZYME PRODUCTION AND ASSAY

AIM: To isolate the protease enzyme producing organism from the environment and assay the enzyme production

PRINCIPLE: Proteolytic enzymes are ubiquitous in occurrence found in all living organisms and are essential for cell growth and differentiation. These enzymes not only play an important role in the cellular metabolic processes also gained considerable attention in the industrial community. Commercial proteases are mostly produced from various bacteria. Culture conditions play significant role on growth and production of protease by bacteria. Based on their acid-base behaviour, proteases are classified into three groups i.e. acid, neutral and alkaline proteases. The majority of commercial alkaline proteases are produced by bacteria, especially Bacillus sp. Several Bacillus species involved in protease production are B. cereus, B. sterothermophilus, B. mojavensis, B. megaterium and B. subtilis. Identification and characterization of microbial proteases are pre-requisite for understanding their role in pathogenesis. Proteases are also useful and important components in biopharmaceutical products. Currently, Protease performs numerous varieties of activities in detergent, food, pharmaceutical, leather, laundry, food processing etc.

MATERIALS REQUIRED:

Media and reagents: Bacterial or Fungal culture, Skim milk agar, Casein agar, SD Agar, Potato Dextrose Agar, Lacto phenol cotton blue &Gram’s staining kit, Folin’s reagent, IMViC Media, TSI Agar, phosphate buffer, 1% casein solution, 20% TCA (trichloroacetic acid).

Glassware: Petri plates, Conical flasks, Pipettes, Glass slides

Instruments: Spectrophotometer, Microscopes, Centrifuge, Colony counter, Waterbath.

PROCEDURE: (For bacteria)

1.      Sampling: The test soil samples were collected from the different sites of DRNGPSC Campus garden soil with aid of sterile spatula from 4-5 cm depth in to sterile plastic bags.

2.      Soil samples were air dried at room temperature.

 Isolation and purification:

3.       Isolation of bacteria from soil carried out by serial dilution method and isolated bacterial colonies were purified by sub culturing and stored as slants at 4±2⁰ C.

4.      Characterization of bacterial culture Various biochemical tests were performed for the identification and characterization of isolated bacteria viz Gram staining, Catalase test, Casein hydrolysis, IMViC test, Urease test, Nitrate test.

5.      Morphological, cultural, physiological and biochemical properties of the isolated strains were studied according to the methods given in Bergey’s manual of systemic bacteriology.

 Enzymatic study:

6.      Qualitative screening of bacteria (for protease)

The bacteria were streaked on casein hydrolysed medium and plates were incubated at 35±2⁰ C for 24 hours. A clear zone around the growth indicates proteolytic activity of the strains and visible difference in the extent of zone of clearing was recorded for proteolytic activity

7. Standard curve Prepared different concentration of protein (10¹ - 10¹⁰) in different test tubes by taking 0.1ml of protein and 0.9ml of distilled water and so on.

8. Added 1mlof protein and 1 ml of 1N NaOH in test tube and kept in water bath at 100^o C for 10 min. Tubes were cooled at room temperature and after 10 min add 1ml Folin’s reagent and leave it for 30 min and absorbance was measured at 750 nm.

     9. Quantitative determination (Proteolytic activity): Casein-yeast extract-dextrose broth were used for the enzymatic activity.

    10. Protease Assay The Protease was assayed by 3ml of bacterial supernatant (centrifuged 24hr old culture at 5000 rpm for 20 min) take in sterile test tube add 3ml phosphate buffer and 3ml 1% casein solution and place it on water bath at 35^o C. now add 5ml 20% TCA (trichloroacetic acid) added in reaction.

     11. Content was immediately mixed after adding Folin Ciocalteu Reagent in the mixture after 30 min 6 ml distilled water was added. Now absorbance was measured at 650 nm in every 24 hr till 72 hrs.

PROCEDURE: (For fungi)

1.      Organism and inoculums preparation fungal strains were isolated from soil of DRNGPASC garden soil, Coimbatore, by serial dilution plate method.

2.      Fungus were isolated from 10^-3 - 10^-4 dilutions by plating into Potato Dextrose Agar (PDA) medium. Isolated fungal cultures were screened for protease enzyme production.

3.      The organisms were identified using lacto phenol cotton blue mounting method.

4.      The isolated culture (Aspergillus flavus) was purified by routine sub-culturing and stored at 4^oC for further use.

5.      Enzyme assay by plate zone method:  The activity of alkaline protease was also measured by casein and gelatin plate technique. One unit of enzyme activity was defined as the amount of enzyme in 25µl of enzyme solution that produced a clear zone of 1mm² at pH 8 and 30°C for 18 hours. 25µl - 1mm² 1U 50µl - 20 mm² 10U 100µl - 30 mm² 15U The 10 U for 50µl and 15U of activity for 100µl were achieved by alkaline protease.

Figure: Casein and gelatin was hydrolyzed by alkaline protease showed a clear zone.

Coli phage isolation from sewage and titration

ISOLATION AND TITRATION OF COLI PHAGES

AIM: To isolate and titrate of coli phages from the given sample

INTRODUCTION:

Bacteriophages, they are “bacteria eaters” and are infectious agents that replicate as obligate intracellular parasites in bacteria. A typical phage contains head, neck and a protein tail.  Bacteriophages are classified into two major groups on the basis of their mode of propagation:

1.      Virulent (Lytic phage): Growth of virulent phage in susceptible bacteria destroys the host cells and produces many copies of themselves.  e.g.  T2 and T4 phages of E. coli.

2.      Temperate Phage: phages which are followed in lysogenic cycle.

Plaque assay is one of the widely used approaches for determining the quantity of infectious virus in a sample.  Only viruses that cause visible damage of cells can be assayed in this way.  Plaque assay was first developed to calculate the titers of bacteriophage stocks. Currently, its modified procedure is being used for the determination of titer of many different animal viruses too.

PRINCIPLE: 

When a suspension of an infective phage (e.g. T4 phage) is spread over the lawn of susceptible bacterial cells (e.g. Escherichia coli), the phage attaches the bacterial cell, replicate inside it, and kills it during its lytic release. Lysis of the bacteriophage is indicated by the formation of a zone of clearing or plaque within the lawn of bacteria. In the absence of lytic phage, the bacteria form a confluent lawn of growth.

Each plaque corresponds to the site where a single bacteriophage acted as an infectious unit and initiated its lytic cycle. The spread of infectious phage from the initially infected bacterial cell to the surrounding cells results in the lysis of the bacteria in the vicinity, eventually forming the plaque that is large enough to be visible to the naked eye. Plaques do not continue to spread indefinitely. The size of the plaque formed depends on the virus, the host, and conditions of culture. The number of plaques that develop and the appropriate dilution factors can be used to calculate the number of bacteriophages i.e. plaque forming units (PFU) in a sample. 

Isolation of a Bacteriophage from Sewage Sludge  

1.      Bacteriophages were isolated from sewage samples by using enrichment cultures. A total of five sewage samples were collected from sewage near our college campus.

2. 35.0 ml of a filtered (Prefiltered to remove debris) sample was mixed with 35ml  of 10× Nutrient broth and with 5.0 ml of pure cultures of isolated E.coli. After proper mixing the enrichment cultures were incubated for 24 h at 37°C to allow amplification of lytic Coliphages.

3. 10.0 ml of sewage bacteriophage culture was transferred into a centrifuged tube and the sample was centrifuged at 2000 rpm for 5 minutes.  

4. Most of the remaining cells were pelleted. The supernatant was transferred to a 10.0 ml syringe barrel fitted with a 0.45 micron filter.

5. The supernatant was filtered to remove bacteria from the phage sample. The filtrate (lysate) was stored at 4^oC.

Procedure for Bacteriophage Plaque Assay:

Preparation of Stock Solution by serial dilution

1.      Place six sterile saline tubes (4.5 ml each) in your test-tube rack.

2.      Label one tube “control” and label the remaining five tubes consecutively from 10^-1 through 10^-5.

3.      Label six nutrient agar plates the same as the tubes.

4.      Using a sterile 1 ml pipette, aseptically transfer 0.5 ml of the bacteriophage suspension provided to the saline tube labelled 10^-1.

5.      Mix the tube well by rolling it between the palms of your hands.

6.      With another 1 ml pipette, transfer 0.5 ml from the 10^-1 tube to 10^-2 tube. Mix the tube as in step 5.

7.      Using a fresh pipette for each transfer, transfer 0.5 ml of the suspension from the 10^-2 tube to the 10^-3 tube, and continue this diluting procedure consecutively for the remaining saline tubes. Do not forget to mix each tube well before and after diluting. 

Overlaying Plate with Phage-Agar Mixture 

1.       Obtain six tubes of melted soft overlay agar from the waterbath. Pipette 0.3 ml of a broth culture of E.coli into each of the soft agar tubes. Mix each tube well by rolling between your palms.

2.      Remove one inoculated tube of soft agar from the waterbath. Using a 1 ml pipette, aseptically transfer 0.1 ml of the 10^-1 saline phage dilution into the soft agar tube. Mix the agar tube by rolling it between your hands.

3.      Immediately, aseptically pour the soft agar onto the surface of the nutrient agar plate correspondingly labelled as 10^-1. Replace the lid and without picking up the plate, rotate it gently in a 6-to 8-inch circle on the surface of the table to evenly distribute the agar.

4.      Using a fresh 1 ml pipette each time and working quickly, repeat steps 1 and 2 for the remaining saline phage dilution tubes and for the saline control tube.

5.      For each dilution tube, use its correspondingly labelled nutrient agar plate.

6.      Allow the soft agar to solidify.

7.      Invert and incubate plates at 35°C to 37°C for 24 hours.

Results

1.      After incubation, each plate was examined and the number of plaques was counted on each plate that has clearly differentiated plaques.

2.      The plaques were counted and recorded.

3.      The number of lytic phages was calculated per millilitre that was in the original bacteriophage suspension using formula mentioned above.

Results of Bacteriophage Plaque Assay

If 48 plaques are observed in 10^-5 dilution factor, as the 0.1 ml virus is added, Plaque forming units/ml will be 4.8 X 10⁷. In your practical you can count the plaque forming units, calculate and tabulate is as follows: 

Dilution of phage	10-1	10-2	10-3	10-4	10-5
Number of plaques
Calculations of plaque units/ml