eChapters

Microbiology

Study of microorganisms, a large and diverse group of microscopic organisms, that exist as single cell or cluster. It also includes virus, which are microscopic, but not cellular.

Some cyanobacteria (Blue-green algae) like Nostoc are known to work as one of the two coponenents (photosynthetic partner or phycoboint) of lichens. The second partner of lichen is a lichen fungus or mycobiont. The lichens have not been described as a therapeutic agent in Charak Samhita, the monumental work on materia medica prepared by great Acharya Charak and his group in India more than three thousand years ago. However, these have been used in folklore medicine in many countries of the world and have been regarded to bear antibacterial properties (Katiyar and Katiyar, 2009). They are found in tundra even, the treeless plain of the arctic regions of Russia, Siberia, marshy in summer and frozen hard in winter. It is common to see gunny bags full of lichens for sale in the Masala market near Kesa House, Express Canal Road, at Kanpur. They also comprise a part of meat masala (spices for meat). There are references that Raja (King) Todarmal studied algae in relation to purification of water.

However, the search for antibiotics began in the late 1800s, with the growing acceptance of the germ theory of diseases; a theory which linked bacteria and other microbes to the causation of a variety of ailments. As a result, scientists began to devote time searching for drugs that would kill these disease-causing bacteria.

Introduction

‘IP’ refers to unique, value-adding creations of the human intellect that results from human ingenuity, creativity and inventiveness. An IP right is thus a legal right, which is based on the relevant national law encompassing that particular type of intellectual property right. Such a legal right comes into existence only when the requirements of the relevant IP law are met and, if required; it is granted or registered after following the prescribed procedure under that law. All countries around the world have a national legal system of intellectual property rights which has developed over varying periods of time during the last 150 years or so. It has enabled the grant of property-like rights over such new knowledge and creative expression of mankind, which has made it possible to harness the commercial value of the outputs of human inventiveness and creativity. This is usually done by its orderly use, exchange or sharing it amongst various types of business partners in a complex network of strategic relationships that generally work harmoniously during the new product development process for creating and marketing new and improved goods and services in domestic and export markets.

IPR is a tool for Innovation and reward for it. Innovations are concerned with the commercialization of new ideas; in contrast, an ‘invention’ may not be directly associated with commercialization. As such, innovation may be seen as a process of interaction and feedback during the various stages of the new product development process. An invention is considered as the generation of a new idea or knowledge, which aims to solve a specific technical problem. Inventions could relate to products or processes and are characteristically protected by trade secrets, utility models/petty patents or patents. Utility models/petty patents or patents are granted/registered under the relevant national/regional law by the relevant national or regional patent office. As not all inventions are commercialized, so it is clear that not all inventions result in innovations.

Keeping in view the importance of intellectual property rights and their protection, Journal of Intellectual Property Rights (JIPR) was started in 1996. The objective of the journal is enhancing communication between policy makers, organizational agents, academics, and managers on the critical understanding and research on intellectual property; and to promote the development of the newly cultivated research field. The journal publishes contributed/invited articles, case studies and patent reviews; technical notes on current IPR issues; literature review; world literature on IPR; national and international IPR news, book reviews, and conference reports.

JIPR is covered in the abstracting/ indexing services namely Scopus, EBSCO host Electronic Journals Service, Directory of Open Access Journal, Library & Information Science Abstracts database and Legal information Institute of India database.

Hunter and Mohler discovered the isozymes. In 1959 Markert and Moler introduced the concept of isozymes, which they defined as the different molecular forms in which proteins may exist with the same enzymatic specificity (Buth 1984). This means that different variants on the same enzymes have identical or similar functions and are present in the same individual. As such, their importance for understanding gene action in development and differentiation was exploited during the1960s in both animals and plants. Nevertheless, isozymes played a minor role in research on plant biochemistry until 1966 when genetic polymorphism for isozymes within the same population was discovered (Stebbins 1989, Wendel 1989). That revealed the possibility for population genetics to make precise quantitative estimates of genetic variability based upon one parameter of the molecular structure of the primary products of the genes themselves.

Plant population genetics were not long in following their zoological colleagues and the investigation of both animals and plants increased explosively. This application, however, necessitated a partitioning of the isozyme concept, since the relevant isozyme subset, defined by Prakash (Buth 1984) as the variant proteins produced by allelic forms of the same locus, to avoid the now common confusion with isozymes which are the various polymer produced from monomers specified by different loci. The development of genetic distance or similarity coefficients (Lukasová 1985) allowed the summarization of isozyme data for intersample comparisons and so quantified isozyme data were soon applied to comparative studies of taxa.

Polyhydroxyalkanoates (PHAs) are diverse biopolyesters synthesized by many bacteria as carbon and energy reserved materials under unbalanced growth conditions. Sand-dunes have low nutrient contents especially nitrogen (N) and phosphorus (P) thus could harbor potential PHA accumulating bacteria. There is no report of isolation of PHA producing bacteria from the sand-dunes environment.  Sand samples of rhizosphere and non-rhizosphere regions of the coastal sand-dunes were processed on Nutrient Agar and TYG-Agar media for isolation of heterotrophic bacteria.

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

1. Mud sample
2. 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)
3. Sterilized bottle with plug
4. Fluorescent tube

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

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.

Viruses are smaller than bacteria and require living cell for their growth. They can be detected through the bacteria to which they parasitise. Viruses are associated with both eukaryotes as well as prokaryotes and contain either DNA or RNA; only few have both DNA and RNA.

Viruses that infect bacteria are called bacteriophage. They consist of head or capsid and a tail. The head may be round, oval, polyhedral and is made up of proteins while tail is hollow and provides an exit for nucleic acid to enter in bacterial cell. The tail also helps in attachment to the cell wall surface of bacteria. After injection of the nucleic acid into bacteria they multiply and form large number of bacteriophage particles which cause lysis of the bacterial cell wall releasing many bacteriophages. Phages that cause lysis are known as lytic phages and that do not cause lysis are called temperate phage. The most common lytic phage is E.coli – a coliphage.

Introduction

The isolation of mutants is often critical to the study of cellular process, especially when such studies are performed at the molecular level. The phenotype of a particular mutant is attributable to the loss of a particular cellular function; the loss of that function is actually attributable to a mutation in a gene necessary for that function. In this experiment we will isolate auxotrophic mutant which can grow only if provided with specific amino acid. For example arg^- mutant have lost the catalytic function of an enzyme involved in arginine biosynthesis due to mutation in the structural gene that encodes the enzyme.

Mutagenic Agent

Mutation is an alteration in the coding sequence of a gene. Mutation occur spontaneously at a frequency of about 10^-8 per bacterial cell per generation. The rate of mutation can be increased with mutagenic agents. Mutants are strains with any structural alteration of their DNA that gives rise to a mutant phenotype. Mutagens are chemical agents (e.g. nitorsoguanidine, 5 bromouracil), physical (e.g.UV) and biological agents (e.g. trnasposons) that cause mutation to occur.

1. Bacterial (Prokaryote) Genome

The chromosome of most bacteria is a single super coiled double stranded circular DNA molecule. A complex packaging mechanism is necessary in order to ensure that the entire DNA is folded within the bacterial cell in a manner, which will not inhibit transcription nor allow entanglement of the two daughter strands to occur during replication (Highly condensed >10 mg/ml in region less than l ìm in diameter). This is achieved by two main mechanisms.

• First the DNA is folded into between 40-100 loops
• Second each of the quasicircles is it self super coiled independently of the others each loop are abolished by Ribonuclease.

Deoxyribonucleic remove the super helical nature of the loops, though limited treatment with this enzyme will attack only a small number of the loops and therefore leads to removal of only some of the super coiling. The intact folded chromosome or nucleoid can be isolated in the presence of non ionic detergents and high molarities of salt in two possible forms free and membrane associated. The free form which sediments at 1600-1700s consists of about 60%DNA, 30%RNA and 10%protein the bulk of which is the enzyme RNA polymerase. The membrane bound form contains an additional 20% of membrane-attached protein. Bacterial genome size ranges from 580 to 9900kbp, & the average gene size is constant from 900 to 1100bp of which 87-94% are protein coding (Rickettisa’s 76% and Borelia Plasmid’s 41% are exceptions).

Introduction

Mutation refers to any heritable change in the base sequence of a DNA molecule. A mutagen is a physical of chemical agent that causes mutations to occur (or increase the frequency of their occurrence). Mutagenesis is the process of producing a mutation. If the mutation occurs in nature without the addition of a mutagen, it is referred as a spontaneous mutation, and the resulting mutants are spontaneous mutants. If it caused by a mutagen, the process is induced mutagenesis.

Ultraviolet light is a fairly potent mutagen. The main DNA lesions are two chemically different types of covalent dimers: cyclobutane-pyrimidine dimers and pyrimidine dimers. The pyrimidine dimers only account for 20% of the total dimers but are correspondingly more mutagenic. In E. coli the number of mutants induced by ultraviolet (UV) light can be reduced by exposure to visible light (photoreactivation), which implicates cyclobutane pyrimidine dimers since dimers can not be photoreactivated.

Aromatic amino acid of proteins, and purine as well as and pyrimidine bases of nucleic acid absorb ultra violet radiation. Pyridine bases, particularly thymine of DNA is the main target compound involved in bactericidal action of ultra violet radiation. The energy absorbed by thymine causes it to under go a photochemical reaction with adjacent ( adjoining) thymine molecule in same DNA strand. DNA replication is blocked because the formation of thymine: thymine (T:T) dimmers. As the dosage increase, cytosine-thymine (C:T) to (C:C) cytosine-cytosine dimmers can be found to add the lethality of ultra violate radiation.

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.

By Alkaline Lysis Method

Introduction

DNA of prokaryotic cells is relatively simple in comparison to that of eukaryotic cells. In addition to chromosomal DNA, a bacterium may also carry an additional piece of DNA called a plasmid. Bacterial plasmids are double stranded closed circular DNA molecules that range in size from 1 kb to more than 200kb. Plasmids are useful to bacterial cells since they carry genes for antibody resistance, RM system etc that allow bacteria to survive in non-ideal conditions.

Plasmids constructed in the laboratory are used as cloning vehicles. These synthetic plasmids contain only essential features such as origin of replication, antibiotic resistance, multiple cloning sites etc. various copy number of plasmid For e.g. pBR322 20 copy/cell – low copy number, pUC 18, pUC 18- 800 copy/cell – high copy number.

Alkaline lysis, in combination with the detergent sds, has been used for more than 20 years to isolate plasmid DNA from E.coli (Birnboim and Doly 1979). This experiment describes how to extract plasmid from bacteria – which involves growth of bacterial culture, harvesting and lysis of bacteria and isolation of plasmid DNA from the lysate using alkaline lysis method. The DNA is analysed on agarose gel.

4.1 From Soil

Soil contains varied group of microorganisms such as fungi, bacteria, actinomycetes, algae and protozoa. The type of nutrients available, moisture, temperature and pH of the soil determine the nature of microbial population in soil. Different laboratory techniques for isolation of microorganisms have been detailed in chapter-III. These are used in particular for the isolation of specific type of microorganisms. All the techniques adopted are just to segregate the pathogens/ microorganisms from the mixture of microbial population present in nature.

(a) Isolation of Fungi

Soil contains large number of fungi and constitutes a major place of microbial population. They can be isolated in pure form by following the methods given below:

Introduction

Yeast contain a hard cell wall. Spheroplasts are yeast cells where the cell wall has been enzymatically degraded. These spheroplasts are very fragile so we must be careful with them. Once we have spheroplasts it will be very easy to lyse the yeast cells and quickly release the RNA and proteins in the cell into a controlled environment where we can temporarly keep the cellular RNases from degrading the RNA. We don’t want to lyse the cells before they are in this controlled environment.

Growth of Yeast Cultures

Cultures of wild-type and yeast containing various mutations will be grown up the day of the experiment. Each section will be given one wild type yeast culture and a culture containing one of the mutant varieties that we have discussed in class.

Yeast are grown very similarly to bacteria. Like bacteria, they have different stages of growth. Scientists divide these stages of growth into early-log phase, mid log-phase, late log-phase, and the stationary phase. These phases are based on the amount of yeast that are around in the broth and how quickly they are able to grow and divide based on nutrient use. Reading the absorbance of the sample at 600 nm is a way in which you can determine what phase of the yeast growth series you are in and how many cells you have.

Haploid yeast cells contain approximatley 1.2 pg of RNA per cell

Predict approximatly how much RNA you should isolate

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

1. Yeast Extract Mannitol Agar (YEMA)
2. Sterile saline
3. HgCl₂ solution (1%)
4. Ethanol (70%)
5. Root nodules of Mung bean
6. Water blanks
7. Pipettes
8. Petri plates
9. BOD Incubator

Under natural habitats microorganisms usually grow in mixed populations containing several species. This is really a problem for microbiologists because a single type of microorganism can not be studied adequately in a mixed culture. In laboratories these populations can be separated into pure cultures. The pure cultures contain only one type of organism and are thus suitable for studying the cultural, morphological and biochemical characters of an individual species. In the present days there are several ways of developing pure cultures in laboratories. Some of which are as follows: