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The chief functions of Clinical Microbiological laboratory are to examine and culture specimens for microorganisms, to make accurate species identification of important isolates and to perform antibiotic susceptibility tests when indicated. These tasks will assist physicians in the diagnosis and treatment of infectious diseases.

Once the specimen is received in the laboratory, the specimens are examined visually depending on the physicians order and the nature of the specimen. Wet mounts and smears may be prepared and stained for microscopic examination. Observations may or may not be immediately reported to the physicians depending on the definitiveness of the results. Timely information may often be used to establish a presumptive diagnosis and institute a specific course of therapy. Specimens that require definitive identification of potentially pathogenic microbes are processed further. All agar plates are streaked for colony isolation; then plates and broths. All cultures are placed in an incubator with appropriate temperature and environmental conditions to maximize the growth and replication of microbes. Often a presumptive microbial identificationcan be made. A final report should be delayed while subcultures and additional test procedures are performed to identify the organisms definitively.

Preliminary Identification of Unknown Bacteria
A number of techniques may be used in the direct microscopic examination of clinical specimens to demonstrate the presence of microbes The examination of wet mounts of unstained materials by phase contrast or
dark field microscopy is useful for demonstrating motility, spirochaetes and endospores. Giemsa, Wright or acridine orange stains may be helpful in observing bacterial forms that stain poorly or that have little contrast from
back ground material.

Cell culture is the most convenient and widely used system for the isolation and propagation of many viruses. Either primary cell culture or established cell lines can be used to isolate the suspected viruses. One can normally get 10-100 virus particles per cell inoculated. Many details of the infection process are controllable to a high degree (including cell density, age, multiplicity of infection (M.O.I.), medium composition, cell type and length and temperature of incubation). Many cell types from different species can be susceptible to a particular virus and vice versa. For replication to occur, the cell type used must be permissive for a particular virus under study.

Protoplasts are cells which have had their cell wall removed, usually by digestion with enzymes. Cellulase enzymes digest the cellulose in plant cell walls while pectinase enzymes break down the pectin holding cells together. Once the cell wall has been removed the resulting protoplast is spherical in shape. Digestion is usually carried out after incubation in an osmoticum (a solution of higher concentration than the cell contents which causes the cells to plasmolyse). This makes the cell walls easier to digest. Debris is filtered and/or centrifuged out of the suspension and the protoplasts are then centrifuged to form a pellet. On resuspension the protoplasts can be cultured on media which induce cell division and differentiation.

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.

1. VARIETAL SEED PRODUCTION

3 m : Rice, soybean

5 m : Blackgram, greengram, cowpea, field lab lab lab lab

5-10 m : Groundnut

25 m : Tomato

Alkaloids are a class of basic, naturally occurring organic compounds that contain at least one nitrogen atom. Alkaloids showed quite diverse medicinal properties (Rates, 2001). Many of them possess local anesthetic properties, but their practical use is limited for clinical purposes (Hesse, 2002). Morphine is one of the most known alkaloids isolated from Papaver somniferum (opium poppy), which had been used and still is for medical purposes. This alkaloid is a powerful narcotic which is used for the relief of pain, but its usefulness is limited because of its addictive properties.

The isolation of microbes is commonly done using serial dilution technique.

1. Trichoderma

Trichoderma is one of the important biocontrol agents. The colonies first produce mycelial growth and then turn yellow and finally produce a dark green color mat.

Anthocyanins are members of the flavonoid group of phytochemicals, a group predominant in teas, honey, wines, fruits, vegetables, nuts, olive oil, cocoa and cereals. The primary players in this group include the anthocyanins (eg, cyanidin, pelargonidin, petunidin), flavonols (quercetin, kaempferol), flavones (luteolin, apigenin), flavanones (myricetin, naringin, hesperetin, naringenin), flavan-3-ols (catechin, epicatechin, gallocatechin), and although sometimes classified separately, the isoflavones (genistein, daidzein) (Skibola and Smith, 2000).

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).

ABSTRACT

A plant grows in a highly competitive environment. It is continually being threatened by other plants encroaching upon the space from which it draws its sustenance, by microorganisms, by insects, avian, reptilian or mammalian herbivores. In order to survive, each plant must draw upon a complex of defenses, which may be physical, such as Spines or Leathery Leaves or Chemical. These defenses are passive, coming into action only when the plant is attacked which repel insects or the herbivores that attempt to feed on them. Plants protect themselves from insect attack by providing a multitude of repellent chemicals including toxic and bitter alkaloids and tannins. Non-specialist insects and other animals that attempt to feed on the plant may be repelled, injured through severe gastrointestinal malfunction or even killed. The cabbage, spinach, mustard etc. are well known for their mustard pungent flavor that deters most animals from consuming it. Even most medicinal plants like Cynanchum collialatum of the family Asclepediaceae have tannins or alkaloids like “cardenolides” that repel most animals and insects. All these plants have a basic enzyme “Myrosinase” which react with these specific tannins to produce the toxic metabolite, which repels insects and animals. But caterpillars of few insects are known to tolerate these toxic alkaloids (ex: caterpillar of Striped Tiger Butterfly-Danaus genutia) and in turn sequester the toxic alkaloids into their tissues making themselves non-palatable to their enemies. We hypothesize the analysis of possible roles of toxic alkaloids isolated from these butterflies as bio-insecticides

1. Mitochondria

Mitochondria are known as the “Power House” of a cell because they are the site of ATP production. In addition, they are also involved with photorespiration and oxidation of fatty acids and work in association with peroxisomes, glyoxysomes and chloroplasts.

Isolation of intact mitochondria is rather difficult. This is because the cell walls of plant tissue are rigid as compared to the single-layered wall surrounding the mitochondria. A significant fraction of mitochondria suffer structural damage during the process of tissue homogenization. Therefore, the pelleted mitochondria need further purification by sucrose gradient centrifugation.

Mitochondria can be isolated from green as well as etiolated leaf; however, the latter is preferred to obtain higher yield of mitochondria (Moore and Proudlove, 1983).

12.1. Routine Media for Fungal Isolation

In recent years commercially available readymade culture media have been used in research laboratories for the isolation of fungal plant pathogens. These culture media are standardized and they are in different types. Some media are used for general purposes while some others are used for the isolation and growth of specific pathogens e.g.

Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, and decant supernatant.

Resuspend cells in 400 µL TE by vortexing, add 50 µL 10% SDS, 50 µL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^oC.

Shear DNA by 3-5 passages through a 26 G needle.
Extract twice with 500 µL phenol: chloroform (1:1), and twice with 500 µL chloroform. 
Precipitate nucleic acids by adding 25 µL 5 M NaCl and 1 mL 95% EtOH, vortex, and centrifuge for 10 min, decant supernatant. Dry pellet.

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.

Background
Milk somatic cells are a diverse population of cells found in milk, including epithelial cells, leukocytes (white blood cells), and macrophages. Isolating these cells from milk provides valuable insights into mammary gland health, mastitis detection, and milk quality assessment.
Several methods can be employed for milk somatic cell isolation. Among the others, centrifugation is the most common method of cell separation. Filtration, immunomagnetic separation and flow cytometry are the other methods of isolating specific cell populations.
In bovines, isolating milk somatic cells is useful in milk quality assessment, mastitis detection, and mammary gland biology research. An increased milk somatic cell count level, particularly neutrophil, indicates mammary gland inflammation. Healthy and mastitis glands can be diagnosed by analyzing the differential counting of milk somatic cells.

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.

Background
Peripheral blood mononuclear cells (PBMCs) are a crucial immune system component. Isolating them from whole blood allows for an in-depth study of immune responses, disease mechanisms, and the development of noveltherapies. Lymphocytes, monocytes, and other different  white blood cells (WBCs) possessing circular nuclei make up the cells of PBMCs. These cells can be directly obtained from whole blood using different separating methods. PBMCs can be utilized for multiple purposes, from drug assessment to expression analysis to parentage determination.
• Toxicity assessment of novel compounds: Interaction of drugs with PBMCs can help assess the safety of compounds and set appropriate doses for safe consumption.
• Comparative analysis: In vitro assessment of diseased vs. healthy PBMCs can provide insights into affected biochemical pathways. The effects of foreign chemical compounds can also be assayed to drive future studies.
• Personalized medication: Studying PBMCs can help develop a genetic immune profile, leading to more specificity and effectiveness in personalized medicines.
• Occupational disorders: Diseases developed due to the overexposure of certain toxic compounds can also be determined by testing PBMCs in peripheral blood.

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.

What is dermal fibroblast?

Skin fibroblasts are a type of cell found in the dermis, the middle layer of the skin. They are the most common cells in connective tissue and play a crucial role in maintaining the structural integrity of the skin. Fibroblasts are responsible for synthesizing and secreting extracellular matrix proteins such as collagen, elastin, and fibronectin, which provide support and elasticity to the skin. Additionally, fibroblasts play a role in wound healing by proliferating and migrating to the site of injury, where they aid in tissue repair and scar formation.

Aim: To prepare the primary cell culture of fibroblasts from canine skin.

Tools and instrument required for the procedure

1. Tissue collection box with ice cubes - To prevent contamination and maintain the stable temperature

2. Surgical Gloves - To prevent contamination

3. Tissue collection tube with 1X PBS - To collect sample

4. Laminar flow hood - To work in a sterile environment and prevent contamination

5. Cell culture dish - To digest the skin

6. Sterile 1000 μL and 200 μL pipette and tips-For accurate measurement and transfer of cell suspension and culture media.

7. Forceps - Fine-tipped forceps for handling and manipulating tissue.

8. Scalpel and blade holder - Scalpel for the dissection of skin tissue.

9. Surgical scissors - Chopping of the skin tissue

10. Falcon 70 μm cell strainer - To filter out undigested tissue debris and obtain a homogeneous cell suspension.

11. 15 ml and 50 ml Centrifuge tubes - To collect and centrifuge the digested cell suspension.