As we delve deeper into the fascinating and complex world of Microbiology, we are constantly presented with new challenges and opportunities to better understand the microbial world. Microbiological techniques are essential in unlocking the secrets of microorganisms and determining their role in our world. The development of new and improved Microbiological techniques has opened up a world of possibility for researchers and scientists alike. These methods have provided us with tools to study the activity, expression, and diversity of microorganisms in different environments, from the human body to the depths of the ocean. In this book, I have compiled a comprehensive guide to the most commonly used Microbiological techniques. I have covered a wide range of topics, from basic laboratory techniques to food

1. General Instructions Instructions to work in the laboratory are given below. Students are advised to read them carefully before they begin their work in the laboratory. Apparatus to be used should be neat and clean. Graduated pipette should be used for pipetting. The small amount of liquid remaining in the pipette should not be blown out while standardizing the pipettes; allowance is always made for this. However, a frosted ring near the top of recently manufactured pipettes indicates that the last drop is to be blown out.

Introduction Microbes like all other living beings require suitable nutrients as well as favourable environment like pH, osmotic pressure, temperature, humidity etc. for growth. The substrate/food base which fulfills any of these requirements and supports the growth of an organism is called culture medium. Selection of a medium to grow a particular type of microorganism is extremely important, as the microbes vary in their nutritional requirements. Thus, different media are used for different groups of microbes. The following are the main types of media: a) Natural/Empirical media: These are made up of natural substrates, such as herbaceous or woody stems, seeds, leaves, corn meal, wheat germ, and oatmeal etc.The exact chemical composition of this media is not known. e.g. Nutrient agar b) Semi-synthetic media: 

Introduction Soil is the natural habitat of a number of microbes viz. bacteria, fungi, actinomycetes, algae and many viruses. Out of these, bacteria are morphologically very simple as made up of just one cell of small size, therefore can multiply rapidly. Each viable bacterium at appropriate dilution can develop into a colony. Hence by counting the number of these colonies which develop on a selective growth medium, the number of viable cells of bacteria present in soil can be ascertained. Since the number of the bacteria present in soil is very high, the soil suspension is to be sufficiently diluted to reduce the number of colonies and to prevent their overlapping. The number of colonies formed from a diluted sample multiplied by the extent of dilution (dilution factor) gives the number of viable cells present

Introduction Fungi constitute an important part of microflora of normal soil. In soils, fungal propagules vary from 2 x 104 to 1 x 106 per gram soil. The propagule being considered as fungal hyphae, spore, and hyphal fragments which is capable of giving colony on the plate. Most soil forms have a filamentous organisation capable of reproduction by means of spores. Their development is especially favoured by soils having acidic reactions. Soil fungi has significant role in organic matter decomposition, as a plant pathogens and in the interaction with other soil microorganisms. Agar plate count method is commonly used as a measure of fungal population in soil.

Introduction The actinomycetes are gram positive, non-motile, non-sporulating and nonacid fast bacteria. Sometimes these are called ray fungi and are connecting link between the bacteria and the true fungi. They possess a very fine vegetative mycelium that does not fragment into bacillary or coccoid forms and therefore their colonies on agar are commonly cohesive or tough in consistency. In many soils, the actinomycetes accounts from 10 to 25% of the total count on agar plates. Their number is higher in soils of low moisture content than in wet soils. They are responsible for degrading more resistant carbon material in soil like humus. They are also important flora of manure heaps. Most of these, particularly Streptomyces, produce antibiotics and so are of a great value

Introduction Bacteria are very minute and colourless in their natural environment and are difficult to be seen under compound microscope. To view their exact morphology, various kinds of stains are used. Stains are of three main types. Acidic stains: These are those stains in which charge on dye ion is negative. e.g. India Ink, Eosin etc. Basic stains: These are those stains in which charge on dye ion is positive. e.g. Methylene blue, Crystal violet etc, Neutral stains: These are the salts of dye acid in dye base. e.g. Eosinate of Methylene blue.

Introduction The motile bacteria show movements with the help of flagella. The motility of bacteria can be observed by hanging drop method under microscope. Requirements Bacterial culture Cavity slide Vaseline Coverslip Procedure Place a very small drop of 24 hours old bacterial culture in the center of a coverslip. Put a small amount of Vaseline at each cover of the depression side of the cavity. Invert the cavity slide over the coverslip. The coverslip adheres to glass slide. When the coverslip is inverted, the hanging drop is suspended in the well.

Introduction A haemocytometer is a single piece of glass with an H-shaped trough forming two counting areas. Hemocytometers are used to determine the total cell count and viability of many different cell types The full grid on a hemocytometer contains nine squares, each of which is 1 mm1. Each counting area is defined by a grid system. It has supports that hold the cover glass at the proper distance (0.1 mm) above these areas for the calculation of the concentration per volume of liquid. Requirements Inoculum Inoculation needle Test tubes

Introduction A microscope is an optical instrument consisting of a lens or a combination of lenses for making magnified images of minute objects. A simple microscope consists of a single lens of magnified glass held in a frame, usually adjustable and has a stand for holding the object and a mirror for reflecting the light. But a compound microscope consists of two lenses i.e., objective and eye piece respectively. A compound microscope gives much greater magnification than simple microscope. The compound microscope will be extensively used in this course in studying various morphological and staining characters of soil microorganisms.

Introduction Photosynthetic bacteria are autotrophs and anaerobes. These bacteria produce Hydrogen sulphide. These are predominant in the bottom of stagnant water enriched with mud. Requirements Mud sample Enrichment medium of Photosynthetic bacteria or salt solution (1.0g/lt. of (NH4)2SO4 1 g; K2HPO4, 1 g; MgSO4,0.2g; NaCl, 5g; pH 7.2) Sterilized bottle with plug Fluorescent tube

Introduction Rhizobia are Gram-negative soil bacteria , aerobic nitrogen fixers and adhere to the roots of leguminous plants. Fixed nitrogen is assimilated to proteins and is transported along the plants. A part of proteins is also released in rhizosphere region. Requirements Yeast Extract Mannitol Agar (YEMA) Sterile saline HgCl2 solution (1%) Ethanol (70%) Root nodules of Mung bean Water blanks Pipettes Petri plates BOD Incubator

Introduction As Rhizobia are slow growers so their enumeration by plating techniques is less recommended. Rhizobia can be enumerated in the presence of a large number of other organisms by most probable number (MPN) method. Soil containing Rhizobia is diluted in liquid and dilutions are used to inoculate roots of legumes. The presence of Rhizobia in a given experimental unit, inoculated by a given dilution, is indicated by the formation of at least one nodule on a root. Thus, the method involves the growth of legumes under conditions such that a single rhizobia cell can multiply and forms at least one nodule on the root. Most Probable Number Method: This method permits estimation

Introduction Biological nitrification is a two-phase process, which consists of the oxidation of ammonia-nitrogen to nitrite-nitrogen by species of genera Nitrosomonas, Nitrosospira and Nitrosogloea, and the subsequent oxidation of nitrite-nitrogen to nitrate-nitrogen by species of the genera Nitrobacter and Nitrocystis. The following equations explain the reaction.The importance of nitrifying organisms in soil is based on their capacity to produce nitrate, the major source of nitrogen assimilated by higher plants. The rate of nitrification is dependent on such factors as concentration of NH4+, temperature, moisture, aeration and pH. Bacteria have their optimal activity at an alkaline reaction. Below pH 7.0 the process declines.

Introduction Phosphate solubilising microorganisms include different groups of microorganisms such as bacteria, fungi and actinomycetes, which convert insoluble phosphatic compounds into soluble form. The species of Pseudomonas, Micrococcus, Bacillus, Flavobacterium, Penicillium, Fusarium, Sclerotium, Aspergillus and others have been reported to be active in bioconversion. Such bacteria and fungi can grow in media where Ca3(PO4) 2, FePO4, AlPO4, apatite, bone meal, rock phosphate or similar insoluble phosphate compounds are the sole source of phosphate. Such microorganisms not only assimilate phosphorus but they also cause a large portion of soluble phosphate to be released in quantities in excess of their own requirements. The solubilisation is not restricted to calcium salts but iron

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

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 Swab Nutrient agar Petri plates Bacterial cultures Forceps Antibiotic discs BOD Incubator Procedure Prepare the bacterial lawn on Nutrient agar plate.

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 Nutrient agar medium Card board Incubator Petri plates Inoculating loop Bacterial cultures

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

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

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 Erlenmeyer flasks (100 ml) Culture of E. coli Pipettes Spectrophotometer pH meter Procedure

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 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 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 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 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 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 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 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 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 Sample Crucibles 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 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 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 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 Food sample Mechanical blender Sterilised distilled water Steilised distilled water blanks (9 ml) Petri plates Sterile Pipettes (1 ml) Eiosin Methylene Blue agar medium Incubator Colony counter Marker Procedure Weigh 1 g of well blended

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 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 Dye reduction tests show the comparative activity of the bacteria in milk, therefore are considered as rough indicator of the number of bacteria per millilitre of milk. These tests find wide application in dairy industry especially in grading of milk for manufacturing purposes. The small amount of equipment, materials and simplicity of method permit simultaneous testing of several samples. Since it combines speed and adaptability, this test may be used in milk plants, receiving stations, condensing plants, cheese factories and similar establishments to rapidly grade incoming milk. Principle It is based on the observation that the colour imparted to milk by a small amount of methylene blue disappears in a time, the length of which is dependant largely upon the number of microorganisms

Introduction Resazurin has high tinctorial value (related to colour), imparting characteristic blue colour. Resazurin test gives us indirect information concerning bacterial content and /or activity. Resazurin is also sensitive to the reducing action of leucocytes. The results of this test are comparatively less affected by antibiotic residues in milk than methylene blue reduction test. Principle Reduction of dye is indicated by a gradual colour change through various shades of purple and mauve to full pink, at which point the resazurin completely changes to resorufin.

Introduction Due to inherent composition (high moisture and nutrients) of meat, it is highly perishable in nature. Hence, preservation of meat is essential to maintain its fresh quality. This is accomplished by various preservative techniques based on the principles of temperature control, moisture control and direct microbial inhibition. Temperature Control 1. Chilling/Refrigeration In this method, meat is stored at refrigeration temperature of 2 to 5°C. Chilling of animal carcasses is also carried out during ageing process, cutting transit, retail display and even in consumer household before ultimate use. Relative humidity during chilling is kept about 90%. The fresh meat has shelf life of 5-7 days at refrigerated temperature