eChapters

Introduction

Standard Plate Count (SPC) is used for determination of population of viable microbes. Enumeration procedures are based on the assumption that microbial cells present in a sample mixed with an agar medium form visible separated colonies. The bacterial colonies developed on the plates are counted. Each colony is presumed to have developed from one bacterium or clump of bacterial cells. Therefore, the results are expressed in colony counts per /ml of sample or colony forming units per /ml (cfu /ml). Total number of colonies counted on the plate multiplied by the dilution factor represents the number of viable organisms in a sample. SPC is a useful method for evaluating the microbiological quality at various stages of processing. It is also helpful in detecting the source of contamination in the product.

Introduction

Bacteriological examination of milk indicates the hygienic condition of the milk i.e., quality of milk in relation to the health of consumer. Two main objectives of carrying out bacteriological examination are to evaluate the degree of cleanliness in the vicinity of production and handling of milk; and secondly to check the presence of infectious microorganism. Generally, number of bacteria present per milliliter is taken as an index in the routine examination of milk as other tests for identification and differentiation of pathogenic and non-pathogenic microorganisms are time consuming and expensive.

Direct Microscopic Count (DMC) consists of examining stained films of measured volume of milk or milk product, dried on a glass slide under a compound microscope. This method enables rapid estimation of total population of bacteria of a milk sample with relatively more accuracy as compared to other methods.

Introduction

Both beneficiary and deleterious microorganisms are present in food. Beneficiary microbes are used for the preparation of a number of dairy and meat products and are called Probiotics, whereas deleterious microbes can cause food poisoning. The quantitative analysis of food products is necessary to find out the presence of types of microorganisms.

Requirements

1. Food sample
2. Mechanical blender
3. Sterilised distilled water
4. Steilised distilled water blanks (9 ml)
5. Petri plates
6. Sterile Pipettes (1 ml)
7. Eiosin Methylene Blue agar medium
8. Incubator
9. Colony counter
10. Marker

Procedure

1. Weigh 1 g of well blended

Introduction

In recent years, the boom in industrialization has revolutionised the countries but has also added much of sufferings. There is tremendous increase in the water and air pollution. Large amounts of effluents from industries containing organic substances of different nature are discharged and enter the aquatic systems resulting in water pollution. Biological Oxygen Demand (BOD) is the amount of dissolved oxygen required by the aerobic microflora to oxidise the organic matter of dirty water. Increased organic matter results in more oxygen requirements by microorganisms to oxidise it and it results in enhancement of BOD of water. As the BOD of water rises, due to decrease in dissolved oxygen (DO), there is increase in death of fish due to suffocation. Determination of biological oxygen demand (BOD) only is insufficient to give a clear picture of organic content of water bodies and the presence of toxic chemicals in the sample so the chemical oxygen

Introduction

Sugars are commonly found in the form of biopolymers. Cellulose, a biopolymer of glucose is the predominant constituent of plant tissues, and may be shortly described as the structural bases of vegetable world. In this case, the glucose moieties are strung together by P-glycosidase linkages, which form the linear chain, making it resistant to chemical attack. Hemicellulose consists of short, highly branched, chains of sugars. It contains five carbon sugars (usually D-Xylose and L-Arabinose) and six carbon sugars (D-Galactose, D-Glucose and D-Mannose) and uronic acid. Its branched nature renders hemicellulose amorphous and relatively easy to hydrolyse to its constituent sugars. Lignin is a biopolymer rich in phenolic components, which provide structural integrity to plants. Acid and neutral detergent fraction methods (ADF and NDF) involve the gravimetric determination of residue previously treated with acid and neutral detergent solutions. These methods determine the insoluble fraction and their individual components. NDF represents the total plant fibre or cell wall including hemicellulose

Introduction

Organic matter in sediment consists of carbon and nutrients in the form of carbohydrates, proteins, fats and nucleic acids. Bacteria quickly eat the less resistant molecules, such as nucleic acids and many of the proteins. Sediment organic matter is derived from plant/animal detritus, bacteria or plankton formed in situ. Total organic carbon refers to the amount of organic matter preserved within sediment.

Materials

1. Sample
2. Crucibles
3. Furnace

Procedure

Take 1.0 g of sample in a crucible whose empty weight is already recorded. Place this crucible in a furnace. Switch on the furnace and set the temperature at 550°C. Keep the samples for 3 hours

Introduction

The balance between soil organic matter (SOM) levels and nutrient cycling are the key to sustained productivity of agricultural system. Both are closely related through microbially driven mobilisation/immobilisation processes. Three major models have been recognised for C pool viz. Labile C, non-labile C and recalcitrant. The models have been characterised on the bases of their half-life period. The labile C includes oxidisable C (with KMnO4), microbial biomass C (soil carbohydrates) and soil fauna exudates (polysaccharides). Its half-life period is from a few hours to a few days. The non-labile C includes aggregate C and its turn over time is from a few months to a few years while the recalcitrant (stabilised C) includes humic fractions and charcoal C having the turn over time of hundred years. Thus fractionation of SOM is carried out on the bases of humic substances, dissolved organic C, natural C13 abundance, microbial biomass C and ease of oxidation of C. The latter method provides qualitative characterisation of soil C.

Introduction

The soil microbial biomass is an important component of the soil organic matter that regulates the transformation and storage of nutrients. It is a labile component of the soil organic fraction containing 1 to 3% of the soil carbon and up to 5% of the total soil N. The microbiologically mediated processes affect ecosystem functions associated with nutrient cycling, soil fertility, global C change and soil organic matter turnover. The size and activity of the soil microbial biomass must be assessed to fully understand nutrient flux in managed and natural ecosystems.

Soil microbial biomass estimations are useful in investigations that compare temporal nutrient fluctuations along natural and perturbed gradients. The effect of tillage, crop rotations and soil type on organic carbon and nutrient turnover can be assessed by following nutrient pools and activity associated with the soil microbial biomass.

Introduction

Demonstration of biological nitrogen fixation is usually accomplished by estimating the total nitrogen through use of some modification of the Kjeldahl procedure. This technique is accurate and reliable whenever a medium initially low in nitrogen is employed.

Principle

The dried organism is digested with sulphuric acid. Organic and nitrate nitrogen is converted to ammonium sulphate and the ammonia gas is distilled into boric acid forming ammonium borate making weak boric acid alkaline. The ammonium borate is back titrated with standard sulphuric acid until the reaction mixture changes its colour from green to pink due to formation of ammonium sulphate. The standard acid used in back titration of ammonium borate is a measure of nitrogen content of the organism.

Introduction

The major biogenic gases, which contribute to global warming, are carbon dioxide, methane and nitrous oxide. Out of these, methane is the most important gas because its thermal absorption is 30 times greater than carbon dioxide, though its atmospheric concentration is about 200 times less than carbon dioxide. Atmospheric measurements of greenhouse gases indicate that methane concentration had increased by 1 per cent annually to 1.7 ppm (by volume) during the last decade. The steady increase of atmospheric methane concentration in the past 200 years has attracted much attention.

Flooded rice soils provide an optimum environment for methane production and emission. Flooding a soil creates essential redox potential, anaerobic decomposition of organic matter and stabilizes the soil pH near neutral, thus makes the congenial environment for methane emission.

Rice is the world’s most important food crop

Introduction

The enzyme nitrogenase is produced in all N₂-fixing systems. The activity of nitrogenase enzyme relates to the biological nitrogen fixation, which can be determined directly by measurement of fixation product, ammonia and the further products of its assimilation. The activity can also be measured indirectly by measuring the ability of nitrogenase to reduce the number of substrates other than N₂. Substrates such as nitrous oxide (N₂O), azide ion (N₃^- ), cyanide (CN^-) and isocyanide (NC^-) are reduced and the requirements for ATP and reductant are similar to those of N₂ fixation. The reduction of protons to molecular hydrogen has been used to measure the nitrogenase activity.

Dilworth (1966) and Schollhorn and Burris (1966&1967) observed the inhibitory effect of Acetylene (C₂H₂) on N₂ fixation by extracts of Clostridium pasteurianum. Dilworth (1966) further observed by mass spectrometry that the C₂H₂ was reduced to C₂H₄. Both gases are readily measured at very low concentrations by gas chromatographic techniques.

Introduction

All biological reactions in soil are catalyzed by enzymes. Soil enzyme activities are believed to indicate the extent of specific processes in soil and in some cases, can act as indicators of soil fertility. Biological oxidation of organic compounds is generally a dehydrogenation process and there are many dehydrogenases that carry out the following reaction:

XH₂ is an organic compound and is a hydrogen donor and A is a hydrogen acceptor. These systems are integral part of the microorganisms, so dehydrogenase activity can be correlated with active population.

Principle

Measurement of dehydrogenase activity involves determination of Triphenyl formazan (TPF) produced by the reduction of Triphenyl tetrazolium chloride (TTC). TTC is a redox indicator, a yellow coloured water soluble salt which possesses the property of being easily transformed into, methanol soluble formazan (intensely coloured, water insoluble) by reduction. 

Introduction

Urea is one of the common fertilizers used as a source of nitrogen. It has nitrogen in amide form (-NH₂) and this form of nitrogen is not utilized by plants. So it has to be hydrolysed. The enzyme, urease hydrolyses the urea to ammonia and ammoniacal nitrogen is taken up by plants or further nitrified.

The measurement of activity of this enzyme in soil is important from soil fertility point of view. There are two methods for determination of the urease activity of soils.

• First involves the measurement of end products of urea hydrolysis. i.e., NH₃ or CO₂.

Introduction

Various organic orthophosphoric mono-and di-esters in soils are hydrolyzed when incubated with toluene-treated or irradiated soils, indicative of phosphatase activity. Assays have been identified, based on the rates of release of orthophosphate. Phosphatase activities of field soils showed little or no variation with season but were influenced by the cropping system and nature of the plant cover. Activities generally increased after the addition of inorganic and organic fertilizers to soils. However, inhibition of phosphatase by phosphatic fertilizers and by liming of soils (application of ca and Mg) has been reported. Phosphatase activities were directly related to the organic-P of soils but were not consistently related to available-P contents.

Phosphatases have been used to describe a broad group of enzymes that catalyze the hydrolysis of both esters and anhydrides. These are classified into five categories

Introduction

The pH (hydrogen ion concentration) of the environment provides the maximum effect on the growth of bacteria. Each organism grows at its optimal pH. Due to variation in other factors such as temperature, osmotic pressure etc marked changes in pH have also been observed.

Requirements

1. Erlenmeyer flasks (100 ml)
2. Culture of E. coli
3. Pipettes
4. Spectrophotometer
5. pH meter

Procedure

Introduction

The water potential plays an important role on growth of bacteria. If the medium has higher concentration of solute, it is said to be hypertonic. Due to this, the cytoplasm dehydrates and shrinks, resulting in inhibition of growth of bacteria. If solute concentration is less, the medium is hypotonic and hence maximum osmotic pressure on the cell.

Requirements

1. Cultures of E.coli and Bacillus
2. Nutrient agar
3. Sodium Chloride (stock solution at 5 M concentration)
4. Inoculating loop
5. BOD incubator

Procedure

Introduction

The most important factor that influences the activity of bacterial enzymes is temperature. Most ofthe organisms grow under mesophilic conditions (at25⁰C). If the optimum temperature is increased, the rate of growth of organism is affected due to changes in protein structure which results denaturation of critical cell protein and ultimately inhibitsthe growth. If temperature is below critical temperature, then there is complete stoppage of growth as less activation energy is available for critical process to occur. However, there are certain organisms which can tolerate extreme temperatures viz.

1. Psychrophiles: Tolerate and grow at > 20⁰C temperature
2. Mesophiles: Tolerate and grow at between 20⁰C and 45⁰C temperature
3. Thermophiles: Tolerate and grow at <45⁰C temperature.

Introduction

Most of the bacteria, excepting the photosynthetic bacteria, are damaged by ultraviolet (UV) radiation. UV rays have lower energy content and are capable of producing lethal effect in cells exposed to wavelengths in the range of 200 nm to 300 nm. Nucleic acids absorb these radiations leading to formation of thymine dimers which distort the structure of DNA. Distortion in DNA brings the interferences in DNA replication and transcription. The germicidal effect of UV can be used to determine the minimum amount of exposure required to produce 100% mortality of micro-organisms.

Requirements

1. Nutrient agar medium
2. Card board
3. Incubator
4. Petri plates
5. Inoculating loop
6. Bacterial cultures

Introduction

Antibiotics are the substances produced by one microorganism and are required to inhibit the growth of other microorganisms. Antibiotics are used to combat infectious diseases. The antibiotic sensitivity is very significant due to development of resistance among various microorganisms.

Requirements

1. Swab
2. Nutrient agar
3. Petri plates
4. Bacterial cultures
5. Forceps
6. Antibiotic discs
7. BOD Incubator

Procedure

1. Prepare the bacterial lawn on Nutrient agar plate.

Introduction

Mycorrhizal fungi are ubiquitous in soils throughout the world and form symbiotic relationships with the roots of most terrestrial plants. The infection of roots of most flowering plants by symbiotic fungi and the transformation of such roots into unique morphological structures are called Mycorrhiza. Over the years it has been convincingly demonstrated that inoculation of agricultural crop plants and forest trees with mycorrhizal fungi can significantly stimulate their growth and development. Such probiotic effects are more pronounced in nutritionally poor soils which occur in very large areas of the arid and semiarid tropics.

Mycorrhiza are classified into three different classes