What is Glomerulonephritis?

Definition: Glomerulonephritis (GN) is an inflammatory condition affecting the glomeruli, the tiny filtering units within the kidneys. These filters are essential for removing waste products, excess fluids, and electrolytes from the blood to produce urine. When the glomeruli become inflamed and scarred, their ability to perform this function deteriorates, leading to a buildup of waste and fluid in the body.

What are the Causes of glomerulonephritis?

1. Infections:
   • Viral Infections: Certain viruses can trigger GN. Notable examples include HIV, hepatitis B, and hepatitis C.
   • Bacterial Infections: Bacteria that cause common throat and skin infections, such as Streptococcus spp or Staphylococcus spp
2. Autoimmune Diseases:
   • Lupus: Systemic lupus erythematosus (SLE) can lead to kidney inflammation as part of a broader autoimmune response.
   • IgA Nephropathy (Berger Disease): This condition involves the buildup of immunoglobulin A (IgA) in the glomeruli, causing inflammation and damage.
3. Genetic Conditions:
   • Alport Syndrome: An inherited disorder that not only causes kidney disease but also affects hearing and vision.
4. Toxins and Medications:
   • Certain drugs or exposure to toxins can damage the glomeruli and trigger GN.

What are the symptoms of glomerulonephtitis?

• Early Symptoms: Often, the kidneys may be significantly damaged before symptoms become apparent. Common early symptoms include:
  • Fatigue: Persistent tiredness that can be a sign of kidney dysfunction.
  • High Blood Pressure: Elevated blood pressure often accompanies GN.
  • Swelling: Accumulation of fluid leading to swelling in the face, hands, feet, and abdomen.
  • Blood and Protein in Urine: Known as hematuria and proteinuria, respectively. These indicators can be detected through urine tests.
• Progressive Symptoms:
  • Decreased Urine Output: Reduced production of urine may signal worsening kidney function.
  • Nausea and Vomiting: These symptoms can occur due to toxin buildup in the body.
  • Fever and Flu-like Symptoms: Systemic inflammation can cause these general symptoms.

How to diagnosis glomerulonephritis?

1. Medical History and Physical Exam: Initial assessment to understand symptoms and possible underlying conditions.
2. Urinalysis: Examines urine for the presence of red and white blood cells, protein, and signs of infection.
3. Blood Tests: Measures levels of waste products like creatinine and urea to evaluate how well the kidneys are filtering blood.
4. Ultrasound: Uses high-frequency sound waves to create images of the kidneys. It helps to assess kidney size, shape, and blood flow, and to identify any abnormalities or blockages.
5. Kidney Biopsy: A definitive diagnostic procedure where a small tissue sample is removed from the kidney and examined under a microscope to detect inflammation, scarring, and other pathological changes.

How to treat glomerulonephritis?

1. Management of Mild Cases:
   • Monitoring: Regular follow-ups to track kidney function and symptoms.
   • Lifestyle Adjustments: Includes dietary changes to reduce salt, protein, and potassium intake.
2. Treatment for Severe Cases:
   • Medications:
     • Blood Pressure Medicines: ACE (angiotensin-converting enzyme) inhibitors help protect kidney function by managing blood pressure.
     • Corticosteroids: These drugs reduce inflammation and prevent scarring of the glomeruli.
     • Diuretics: Help to manage fluid retention by increasing urine production.
   • Dietary Modifications: Limiting protein, sodium, and potassium to reduce kidney strain.
   • Advanced Treatments:
     • Dialysis: An external process that filters blood when the kidneys can no longer perform this function.
     • Kidney Transplant: Replaces the diseased kidney with a healthy kidney from a donor, often considered when kidney function declines to end-stage renal disease (ESRD).

What are the main complications of glomerulonephritis?

• Kidney Failure: Progressive GN can lead to kidney failure, requiring dialysis or a transplant.
• High Blood Pressure: Persistent elevated blood pressure may develop or worsen.
• High Cholesterol: Elevated cholesterol levels are common in chronic kidney conditions.
• Blood Clots: Increased risk of clots such as deep vein thrombosis (DVT) or pulmonary embolism.
• Damage to Other Organs: Prolonged GN can affect other body systems.

When to Contact a Healthcare Provider:

• Seek medical advice if symptoms worsen, new symptoms develop, or if there are concerns about kidney function.

Key Points:

Glomerulonephritis is an inflammatory disease of the kidneys’ filtering units, leading to impaired waste removal and potential complications. Early diagnosis and treatment are crucial for managing symptoms, preventing progression, and addressing potential kidney failure. Regular monitoring and treatment adjustments can significantly impact the management of this condition.

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• Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia resulting from impaired insulin secretion

Classification of Diabetes

I. Type 1 Diabetes

1. Insulin- dependent diabetes mellitus
2. Juvenile- onset diabetes

Characteristics of Type I Diabetes

• Hypoinsulinemia
• 10% of diabetes case
• Patient require insulin
• Age onset is childhood
• Ketoacidosis

Etiology of Type I Diabetes

• Autoimmune disease
• Selective destruction of beta cells by T cells
• Several circulating antibodies against beta cells
• Cause of autoimmune attack
• Both genetic and environmental factor are important

II. Type 2 Diabetes

• Non- insulin- dependent diabetes mellitus
  Adult- onset diabetes

Characteristics of Type 2 Diabetes

• Impaired insulin action
• Insulin secretion is normal or increased
• 90% 0f diabetes cases
• Age of onset is adulthood
• Associated with obesity
• Ketoacidosis but rare
• Most cases don’t require insulin

Etiology of Type 2 Diabetes

• Response to insulin is decreased
• The mechanism of insulin resistance is unclear
• Both genetic and environmental factor are responsible
• Post insulin receptor defect

Mechanism of hyperglycemia in diabetes

1. Increase in hepatic glucose output

• Decrease insulin secretion in Liver
• Decrease homeostatic effect on glucagon secretion resulting in increased glucagon
• Gluconeogenesis and glycogenolysis occurs in Liver
• Results in increased plasma glucose

2. Decrease in uptake of glucose in Muscles

• Decrease in insulin in muscles
• Decreased uptake of glucose and amino-acids in muscles
• Increased breakdown of proteins
• Results in increased plasma glucose and plasma amino acids

3. Decrease in uptake of glucose in adipose tissue

• Decrease in insulin
• Increased lipolysis and decreased lipogenesis
• Results in increased plasma fatty acids

Sign and symptoms of Diabetes

• thirst and frequent drinking
• most frequent urination particularly at night
• unexplained weight loss
• fatigue
• blurred vision
• frequent infection of skin, genital

Diagnostic evaluation of Diabetes

• history taking
• physical examination
• symptoms + random plasma glucose > 11.1 Mm (200mg/ dl )
• fasting plasma glucose >7Mm (126mg/dl)
• oral glucose tolerance test (OGTT) 2 hour plasma glucose >11.1Mm (200mg/dl)

Management of Diabetes mellitus

1. Medical management of Diabetes

Type 1 Diabets

• insulin injection should be given as per needed by analyzing blood sugar level
• frequent blood sugar check
• carbohydrate counting should be done

Type 2 Diabetes

• 7,5 % monotherapy ( metformin unless contraindicated)
• 5 – 9,0% dual therapy ( metformin + other medication)
• If the patient have diabetic complication then we must go for insulin therapy
• As Metformin is contraindicated in-case of renal failure, liver, or lung disease

2. Surgical management of Diabetes

• Gastric bypass and biliopancreatic diversion
• Pancreatic transplantation
• Islet cell transplantation

3. Nursing management of Diabetes

1. Nursing assessment of Diabetes A

• History taking, history of family and past medical history
• Past surgical history and other treatment
• Information about medication and insulin therapy
• Assessment of nutritional status
• Assess the blood sugar level
• Assess for sign and symptoms

2. Nursing diagnosis of Diabetes

• Imbalanced nutrition less than body requirement related to reduction of carbohydrate metabolism due to insulin deficiency
• Fluid volume deficit related to polyuria, decreased fluid intake
• Impaired skin integrity related to decreased sensory sensation, impaired circulation
• Risk for infection related to high glucose level reduction in leukocyte function
• Deficit knowledge about the disease the process related to lack of information

3. Nursing intervention of Diabetes

• Monitor vitals sign of the patient
• Provide medication as per cardex , administer insulin or an oral anti diabetic drug
• Analysis blood glucose level
• Maintain fluid and electrolyte balance
• Increase knowledge about diabetes management
• Monitoring and managing potential cmplications
• Provide skin care especially to the feet and legs
• Assist the client for coping mechanism
• Teach patient self- care and about disease condition

Complications of Diabetes mellitus

1. Acute complications of Diabetes

• Glucosuria ( glucose appear in urine)
• Polyuria( frequent urination)
• Polydipsia( excessive thirst)
• Polyphagia(excessive food intake)
• Ketoacidosis

2. Chronic complications of Diabetes

• Neuropathy ( loss of sensation due to damage of nerve fibres )
• Retinopathy ( damage of retina)
• Cataract ( damage of lens)
• In cardiovascular there may be atherosclerosis, hypertension, myocardial infraction
• In nephropathy there may be severe kidney failure and follow- up proteinuria

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•  Pheromones are chemical messengers secreted or discharged outside the body of the organism that activates a social response in members of the same species.
• The term “Pheromone” (Phero-to carry and hormone-to stimulate) was coined by Peter Karlson and Martin Lüscher in 1959.
• They are ectohormones in nature.
• Both plants and animals can release pheromones
• First sex pheromone was identified in 1959 from silk moth (Bombyx mori) termed as bombykol.
• Plants use pheromones to attract bees and other pollinators to their flowers.
• Some plant pheromones have alike chemistry to animal pheromones.
• Aphrodisiacs are fungi that has an odour nearly alike to androstenol; a sex attractant for pigs and very similar to chemicals that act as sex attractants in humans.
•  Both intraspecific and interspecific signals influence many forms of insect behaviour.
•  Chemicals engaged in signalling between organisms and affecting behaviour alteration are called semiochemicals.

These are of 2 major types of pheromones:

1)Pheromones – conciliate intraspecific interactions.

2)Allelochemicals – conciliate interspecific interactions.

Evolution of pheromone

•  Chemical senses being primitive, are shared by all organisms including bacteria, so animals are pre-adapted to determine chemical signals in the environment.
•  Pheromones originate from compounds that originally having other uses or importance.
•   Chemical molecules became signal molecules by increasing sensitivity and specificity.
•   Signals are acquired from movements, body parts or molecules already in use and are eventually changed in the course of evolution to intensify their signal
  function.
•   Evolution in the senses and response of the receiver which facilitated pheromones to become the mostly acceptable way of communication among animals.

Types of pheromones

1. Aggregation pheromones:

•  A group of individuals existing at one location is termed as aggregation.
• Released by male and utilized by species with long-lived adults.
•   These pheromones function in many ways including mate selection, protection against predators, and conquering host resistance by mass attack.
•  Ex: boll weevil (Anthonomus grandis B.) is a oligophagous insect which feeds primarily on cotton, Gossypium hirsutum L. Male boll weevils locate their host plant, feeding ensues and releases aggregation pheromones, grandlure.
•  Male or grandlure baited traps have been used for mass trapping the boll weevil for many years. It is the most ecologically selective pest suppression methods as they are nontoxic and effective at very low concentrations.

           2) Alarm pheromones:

• Some species are capable of releasing a volatile substance in response to attack of the predator which alerts/triggers other members of same species of the danger.
• Ex: Aphides belonging to Homoptera species secrete (E)- β – farnesene as alarm pheromone. These chemicals are released in air as envoy to help other to escape from danger.
• Certain plants emit alarm pheromones when grazed upon, resulting in tannin production in neighbouring plants. These tannins make the plants less beguiling for the herbivore.

           3) Releaser pheromones:

•  Releaser pheromone has an immediate impact on the behaviour of the recipient and alters it.
•  This type of pheromone shows a swift response, but is degraded in no time.
•   Ex: Some organisms use strong attractant molecules to lure mates from a distance of two miles or more.

4) Signal pheromones:

•  Signal pheromones results short-term changes, such as the neurotransmitter release that activates a response.
• For example, GnRH molecule act as a neurotransmitters in rats to gain lordosis behaviour (inward curve of the spine).

            5) Primer pheromones:

• Triggers a chain of physiological development events that may take days to weeks before an unconcealed response is noticed.
• For example- primer pheromones include stimulation of sperm production in fish, termites cast determination, development rates of locust, menstrual cycles in human and other mammals.

6) Epideictic pheromones (Ovipositor pheromones):

•   Also termed as spacing pheromones.
•  They are known to repel, rather than attract.
•  Capable of regulating population density.
•   For examples- in insects, female who lay their eggs in fruits deposit these unknown substance in the territory of the clutch to signal to other females of the same species they should clutch elsewhere.

7) Territorial pheromones:

•  These pheromones explains claimed region of specific organism.
• alert other organisms of nearby dominant animals.
• These are helpful to filter other animals, such as an ant from another colony.
•  Ex: Dogs deposit territorial pheromones present in their urine on landmarks to mark the boundaries their area.

8) Trail pheromones:

•   Recruited by social insects for direction and to employ nest mates to a suitable food source.
•  These are mostly vaporous compounds.
• Ex: When species of wasps such as Polybia sericea found new nests, they use pheromones to lead the rest of the colony to the new nesting site.

9) Sex pheromones:

•   Particularly related with signalling mating behaviours or dominance.
• These are excreted by an organism to lure an individual of the opposite sex and inspire them to mate with them.
•  Generally released by females.
• These are simple and volatile, long chain unsaturated alcohol, acids, benzene derivatives, or bicyclic aliphatic compounds.
•   Ex: The female Bombyx mori (silk moth) secretes bombykol, the first sex attractant isolated from natural source. It is released in air to attract the male from distance. The male organ of B.mori is intensely sensitive of bombykol.

Application of Pheromones in Pest management

• Inspecting a population of insects to determine if they are present or absent in an area.
• To detect if enough insects are present to permit a costly treatment.
• To mass trap insects to eliminate large numbers of insects from the feeding and breeding population.
• Example: Relatives of bark beetles called ambrosia beetles have been mass trapped from log sorting and timber processing areas throughout British Columbia. Disruption of mating in populations of insects. Useful in protecting crops and residents. Synthetic pheromone is dispersed into crops and the false odour plumes attract males away from females that are waiting to mate.
• Push-pull, attract and kill are direct plans for pest killing.
• Mating disturbance by creating hindrances can control pests as well.
• The theory of behavioural manipulation can be applicable to lure the natural enemies of pests  and  increase  biological  control  services in  managed agroecosystems.

References:

• https://www.researchgate.net/publication/267512282_Use_of_Pheromones_in_Insect_Pest_Management_with_Special_Attention_to_Weevil_Pheromones
• https://www.grains.k-state.edu/spirel/docs/research/domestic-presentation/heat-workshop-1/12_Jeff%20Weier.pdf

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Introduction:

• DNA Microarray is one of the molecular detection techniques which is a collection of microscopic characteristics (commonly DNA) affixed to a solid surface.
• Also termed as DNA chip or biochip.
• DNA spots can be probed with the target molecules to result either qualitative or quantitative data.
• Microarrays can be characterized on the basis of the nature of the probe, the solid support used and the specific technique used for target detection and/or probe addressing

Approaches of DNA microarrays

Some of the approaches of microarrays are;

1. Printed microarrays
2. In-situ synthesized microarrays
3. High-density bead arrays
4. Electronic microarrays
5. Suspension bead microarrays

1. Printed Microarrays:

• These were first microarrays to be used in research laboratories.
• It is termed as ‘printed microarrays’ because of the printing or spotting of probes on the microarray surface (generally a glass slide).
• The probe spots can be put in either by contact or noncontact printing.
• The direct application of probe solution onto the microarray surface is performed by print pin in contact printing whereas the noncontact printer uses the same technology as the computer printers to eject the droplets of probe solution onto the glass slide.
• Few nanolitres of probe solution per spot is applied to create an array of 100-150mm features, in both the cases of printed microarrays.
• During the course of printing, it is important to control for cross-contamination and conserve the wholeness of the microarray and following hybridization data.
• On account of comparatively large size of the characters, printed microarrays are lower in density(10,000-30,000 characters) than high density bead arrays and in situ-synthesized microarrays but provide more features than suspension bead arrays or electronic microarrays.
• Printed are comparatively simple and economic than in situ  synthesized microarrays. However, the initial step of microarray facilities is expensive and requires separate space where environmental conditions such as humidity, dust, and temperature are strictly controlled.
• A major merit of printed microarrays is flexibility. In clinical     microbiology, printed microarrays are used due to the ability to adjust spotted probes based upon revised annotations or the finding of noble, rising pathogens.
• However, its use is convoluted by the tedious and expensive tasks of observing manufacture reproducibility, performing clinical validation studies, and continually evaluating the quality of downstream data.
• Even if printed microarrays are favourable to user-defined testing, their use in diagnostic microbiology remains confined to specific research applications.

On the basis of nature of the probes, printed microarrays can be classified as:

1. Double stranded DNA microarrays:

• The probes contains of amplicons acquired by PCR using the primers designed either from a familiar genomic sequence, or cDNA or shot-gun library clones.
• The denaturation of the double stranded amplicons takes place either in print buffer or after immobilization, permitting the probes to be available for hybridization.
• The attachment of the amplicons to the glass slide is favoured by the electrostatic interaction of the negative charge bore by the phosphate backbone of the DNA with the positive charge of the coating of the glass slide surface. It can also take place by the formation of UV-cross-linked covalent bonds between the thymidine bases in the DNA and amine groups on slides.
• PCR amplicons are ideally required to have high specificity and yield eliminating contamination, including nonspecific amplification and taints that affect attachment to the microarray surface. But, dsDNA probes have high sensitivity but lacks in specificity. Less specificity can be advantageous when analyzing a genomic sequence rich in natural polymorphisms.
• A great demerit in the production of printed dsDNA microarray is the massive scale of amplicon production and related troubles of quality control, information management, efficiency and preciseness.

2. Oligo-nucleotide microarrays:

• In this type, the spotted probes contains of short, chemically synthesized sequences.
• The probe’s length ranges from 25 to 80bp but it may be as long as 150bp for gene expression microarrays.
• Shorter probe lengths allows less errors during probe synthesis and enables the interrogation of small genomic regions, plus polymorphisms. Specificity is greater when shorter and specific genomic regions are interrogated. With increasing length of the probe, the strength of the hybridization signal and the sensitivity increases.
• In order to improve the hybridization signal strength, the higher concentration of the probes can be applied during printing.
• Despite being easier to produce than dsDNA probes, oligonucleotide probes need to be carefully designed so that all probes acquire similar melting temperatures (within 5⁰ C) and eliminate palindromic sequences.
• The probe’s attachment to the glass slides takes place by the covalent linkage as electrostatic immobilization and cross-linking can result in significant loss of probes during wash steps due to their small size. The coupling of probes to the microarray surface takes place via modified 5′ to 3′ ends on coated slides that provide functional groups (epoxy or aldehyde)

2. In-situ synthesized Arrays:

• In-situ synthesized arrays are immensely-high-density microarrays which use oligonucleotide probes, (out of which Gene chips are most extensively known).
• Contrasting to printed oligonucleotide array, the oligonucleotide probes which is typically 1-2 cm² are synthesized directly on the surface of the microarray.
• As in-situ synthesized probes are usually short(20-25 bp), multiple probes per target are used to enhance statistical accuracy, sensitivity and specificity.
• Normally,11 probes are used per 600 bases being examined.
• The specificity is further increased by use of probe sets. A probe set consists of one perfect-match probe and one mismatch probe that contains a 1bp difference in middle position of probe. Each member of probe set is present in a separate feature, allowing the mismatch probe to behave as a negative control to recognize possible nonspecific cross-hybridization events.
• Recent progresses in Gene Chips consists the use of longer probes, the design of arrays that cross-examine across entire genes or exons, and the execution of multiple self-standing and non-overlapping perfect-match probes in place of classic probe sets.
• Affymetrix Gene Chips classically have >10⁶  characters per microarray depending on the inter-character distance. The probes are produced using semiconductor based photochemical synthesis.
• Synthesis linkers reformed with light-sensitive protecting groups are available on the quartz surface. Thus, the microarray surface is chemically secured from a nucleotide addition.
• When the array surface is exposed to UV light, reactive nucleotides revised with a photolabile protecting group can be joined to increasing oligonucleotide chains.
• Photolithographic masks are used to target specific nucleotides to specific probe sites. Each mask has a designated pattern of windows, which acts as a filter to either transmit or obstruct UV light from specific characters on the chemically preserved microarray surface.
• The areas of the microarray surface that has been exposed to the light will be deprotected and specific nucleotides can be added. The pattern of windows in each mask controls the sequence of nucleotide addition.
• In-situ probe synthesis is thus completed through the cycling of masking, exposure to light, and the addition of either A,T,C or G bases to the growing oligonucleotide.
• Further high density oligonucleotide arrays include those synthesized by Roche NimbleGen and Agilent technologies. These platforms use longer oligonucleotide probes(60-100bp), but NimbleGen and Agilent uses maskless photo-mediated manufacture, and inkjet technology respectively for the synthesis of probes.
• Also, NimbleGen and Agilent platforms allow multi-color hybridization whereas experiments performed with GeneChips are confined to one label.
• Synthesized microarrays relies on commercial synthesis due to complicated nature of chemical synthesis and huge expense required in production and are therefore favourable for user-defined progress.
• The major merits to these systems are the duplicatibility of the production process and the standardization of reagents, instrumentation, and data analysis, all of which are applied to the clinical laboratory.
• Regardless printed or synthesized, oligonucleotide arrays normally permits much cleaner downstream hybridization than amplicon based microarrays.
• Although in-situ synthesized oligonucleotide microarrays are very vigorous systems and have important control measures included, there are recently none with direct diagnostic contagious disease applications that are commercially accessible.

3. High-density bead arrays:

• Bead arrays issues a patterned substrate for the high-density spotting of target nuclei acids alike the printed and in situ-hybridized microarrays.
• Bead arrays rely on 3-mm silica beads that haphazardly self gather onto one of two available substrates: the Sentrix Array Matrix (SAM) or the Sentrix Bead Chip.
• The SAM comprises 96 1.4-mm fibre-optic bundles. Each bundle is a single array containing of 50,000 5-mm light conducting fibers, each of which is chemically engraved to form a microwell for a single bead.
• Upto 1,536 types of bead gather onto each fibre bundle, resulting in 30 beads of each type in the array in case of universal bead array.
• Each SAM permits the inquiry of 96 independent samples.
• Bead chips are more suitable for high density applications such as whole-genome typing.
• Contrast to the known locations of printed and in situ hybridized microarray characters, the beads in Bead arrays haphazardly group to their final location on the array.
• Thus the bead location is mapped which is completed by a decoding process.
• The SAM can be treated using a standard microtiter plate, which makes it manageable to standard automation and high-throughput processing.
• The distance between individual arrays on the 16-sample Bead Chip is similar to that of a standard multichannel pipettor, thereby mediating ease of use.
• Bead arrays can support up to 105 to 106 characters and have built-in repetition.
• Since each manufactured microarray will not be similar, this redundancy is a pivotal experimental control for inter microarray comparative data.
• An additional merit to the peculiarity of each microarray is that altering the bead pattern produces a method to identify spatial bias.
• Although the analysis tools are present for Bead array-specific data analysis, background rectification, and spatial trace recognition have been delaying behind those provided by other microarray manufacturers.
• Bead arrays have been usually applied to DNA methylation studies, gene expression profiling , and SNP genotyping.

4. Electronic microarrays:

• The printed and in situ-synthesized microarrays and Bead arrays explained earlier depend on passive transport for the hybridization of nucleic acids. Dissimilar to it, electronic microarrays utilize active hybridization via electric fields to control nucleic acid transport.
• For the electronic addressing of nucleic acids, microelectronic cartridges use complementary metal oxide semiconductor technology.
• Each Nano Chip cartridge consists of 12 connectors that control 400 individual test sites.
• When a positive current is applied to one or more test sites on the microarray, negatively charged nucleic acids are transported to specific sites, or features.
• The surface of the microarray consists  streptavidin, which forms streptavidin-biotin bonds once electronically addressed biotinylated probes meet their targeted location. The positive current is then eliminated from the active features, and new test sites can be operated by the targeted application of a positive current.
• The microarray is set  for the application of fluorescently labeled target DNA, once the probes have been hybridized at distinct features.
• Usually, target DNA passively hybridizes with the immobilized probes on the microarray but can also be concentrated electronically. Even though addressing the capture probe down first is the most normally used format, amplicon-down and sandwich assays have also been employed.
• Regardless of the addressing format used, if hybridization occurs between the probe and the target DNA, fluorescent reporters will be present at the positive test, which will be detected when the electronic microarray is examined and analysed.
• Electronic microarrays has many merits. For instance, since multiple probes, each with a discrete fluorophore, can be sequentially addressed to the same feature, multiplex detection can be completed at a single test site.
• The flexibility of this platform permits nucleic acids from an individual sample to be hybridized to multiple test sites for the spotting of multiple targets, or nucleic acids from multiple samples can be examined on the same microarray cartridge, decreasing waste.

5. Suspension bead arrays:

•  Dissimilar to the two-dimensional, or planar, arrays explained earlier, suspension bead arrays are three- dimensional arrays which are based on the utilization of microscopic polystyrene spheres (beads) as the solid support and flow cytometry for bead and target spotting.
• Furthermore, they are discrete from the high-density Illumina Bead arrays discussed earlier, in which the beads are placed on fiber- optic strands or silicon slides.
• Suspension-bead-based assays were initially described in 1977 and focused on the detection of antibodies and antigens.
• Multiplexing was initially achieved by using variable-sized microsphere sets for the continuous detection of multiple antibodies. Currently, more vigorous multiplexing is completed using different microsphere sets based on color.
• Red (658-nm radiation) and infra- red (712-nm radiation) fluorochromes are used at different concentrations to fill 5.6-mm microspheres.
• Each bead of the 100-microsphere set has a different red-to-infrared ratio, and thus, each bead has a distinct spectral address.
• Microspheres with a specific spectral address integrated to a specific probe are identical to a character in a planar microarray.
• Once multiple individual microspheres have been incorporated to separate specific probes, a mixture of microspheres can be used to examine extracted and amplified nucleic acids.
• Using a bench-top flow cytometer, the succeeding  detection of a fluorescent reporter that indicates probe-target DNA hybridization is completed.
• An individual file microsphere suspension proceeds by two lasers. A 635-nm laser stimulates the red and infrared fluorochromes infused in the microspheres, which permits the classification of the bead and thus the identity of the probe-target being examined. A 532-nm laser thrills reporter fluorochromes such as R-phycoerythrin and Alexa 532 to determine any hybridization that takes place on the microsphere.
• For nucleic acid detection by suspension bead arrays, several chemistries have been initiated that includes direct DNA hybridization, competitive DNA hybridization, and solution based chemistries with microsphere capture. In direct DNA hybridization, PCR amplicons hybridize directly to probe capture sequences placed on the microspheres.
• Principally, a biotinylated primer used during amplification permits streptavidin–R-phycoerythrin to attach and label hybridized microspheres.
• Competitive DNA hybridization employs unlabeled PCR amplicons and biotinylated competitor oligonucleotides. In difference to the direct hybridization method, competitive DNA hybridization results high fluorescence in the lack of target DNA. When target DNA is available, it binds the tagged competitor DNA, which, in turn, is not present to hybridize to the microsphere, resulting low fluorescence.
• Allele-specific primer extension (ASPE) or target-specific primer extension (TSPE), oligonucleotide ligation assay (OLA), and single-base-chain extension (SBCE) are solution-based chemistries integrated with succeeding microsphere capture.
• By utilizing the natural properties of DNA polymerases and ligases, these chemistries integrate a capture sequence during the solution-based reaction.
• Both ASPE or TSPE and OLA use a capture primer, which consists a unique 5′ sequence succeeded  by a target-specific sequence. In ASPE and/or TSPE, the primer can be extended by DNA polymerase only if target DNA is available to supply the complementary base for the 3′ nucleotide. The tag in ASPE and/or TSPE is facilitated by a biotinylated deoxynucleotide triphosphate. The OLA reaction is ligase dependent. In addition to the capture primer, a biotinylated probe homologous to target DNA is present during an OLA.
• The capture primer and reporter probes can be joined only if target DNA is present in the sample. Used for multiplex SNP detection, SBCE needs independent reactions for each nucleotide query. For every SNP being cross-examined, one probe with a distinct capture sequence is used to assay the possible alleles in separate wells consisting a distinct dideoxynucleoside triphosphate per well. When the capture and target sequences are homologous, a biotinylated dideoxynucleoside triphosphate is integrated, thereby stopping further augmentation.
• The solution-based chemistries explained above all take advantage of universal microspheres with nonspecific capture sequences.
• The first universal sequences used to label microspheres were ZipCode/cZipCode arrest sequences originally used with SBCE in SNP genotyping assays. The 25-bp ZipCode sequences are based on erratic genomic sequences from Mycobacterium tuberculosis.
• A distinct ZipCode sequence is incorporated in the 5′ end of the capture probe used in the chemistries explained above, while microspheres are labeled with the complementary sequence.
• Even though the character density of suspension bead arrays is the least of all the platforms assessed, merits abound that make this platform the most practical for clinical microbiology applications.
• The presence of universal bead sets and their innate flexibility make the progress of user-defined applications practical and relatively economic. Although users must carefully affirm the positive fluorescent threshold for each analyte in the multiplex, user-defined bead-based assays allow experienced users a multitude of clinically relevant applications.
• Significantly, in 2008, Luminex obtained FDA clearance for the first infectious-disease suspension bead array (xTAG RVP), which diagnose 12 respiratory viruses and subtypes. Although analyte-specific reagents (ASRs) also exist, the presence of FDA-cleared products is an important step in getting this technology into less-experienced detective microbiology laboratories.
• However, many built clinical molecular microbiology laboratories depends greatly on real-time PCR, which has less contamination risks.
• In distinction, the opening of postamplification tubes and the following pipetting steps in the workflow of suspension arrays increase the risk for intra- and inter run contamination. Careful deliberation should be taken to control contamination and the reestablishment of postamplification laboratory space in the era of real-time PCR.
• Nevertheless, the relative clarity, strong multiplexing abilities, and cost effectiveness of suspension bead arrays make this platform the most luring for high-throughput nucleic acid diagnosis in clinical contagious disease diagnostics.

References:

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467903/
• http://www.premierbiosoft.com/tech_notes/microarray.html
• https://www.csus.edu/indiv/r/rogersa/bio181/microarrays.pdf
• https://www.csus.edu/indiv/r/rogersa/bio181/microarrays.pdf

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What is extraction of plant?

•Extraction can be defined as the treatment of the plant (or animal tissues) with solvent, where the medicinally active constituents are dissolved and most of the inert particles remain undissolved.
• The process of extraction developed into a distinct area and contributes mainly to the progress of phytochemistry.
• Extraction processes includes maceration, steam or hydro-distillation, pressing, decoction, infusion, percolation and Soxhlet extraction.
• It is difficult to find a production process in the perfume, cosmetic, pharmaceutical, food, bio fuel, or fine chemicals industries, which does not use extraction processes these days.

Why extraction process of plant materials important?

• To siphon the required chemical components from the plant materials for further separation and characterization.
• To isolate an unknown compound responsible for the particular activity.
• To discover bioactive constituents from plant materials.
• It has great impact on the reliability of a natural medicine.
• Significant for the standardization of herbal/ayurvedic products.

Solvent extraction method

• The efficient determination of biologically active compounds from plant material largely depends on the type of solvent used in the extraction process.
• The ruptured cells are integrated in the extracting solvent and the mixture is kept for some time (half an hour to 24 hr) to let the solvent prick all parts of the ruptured cells.

1. • Maceration:

   • The simplest mode of cold extraction process which is suitable mostly for the thermolabile drugs.
   • In a stoppered container, the powdered plant material is taken and soaked with the solvent for a specified period of time.
   • Agitation is performed until the soluble matter dissolves.

2. • Percolation:

   • It is the process in which movement of mass across a porous material takes place where a percolator is generally used.
   • Percolator is a narrow, cone-shaped vessel open at both ends.
   • The plant (Crude) material is taken in a percolation tube plugged with cotton or fitted with a filter and a stopcock and is moistened with an appropriate amount of solvent(esp. menstruum).
   • It is left to stand for approximately 4 hr in a well closed container.
   • Further menstruum is added to create a shallow layer above the mass, and the mixture is permitted to macerate in the closed percolator (for 24 hr)
   • The liquid contained is allowed to drip slowly by opening the outlet of the percolator.
   • Menstruum is added as desired, until the percolate measures about three-quarters of the desired volume of the final product.
   • The marc is then pressed and the obtained liquid is added to the percolate.
   • Marc is inert fibrous and other insoluble material remaining after extraction.

3. • Digestion:

   • It is a type of maceration in which mild heat (40-60oC) is applied.
   • It is used when moderately elevated temperature is acceptable.
   • It can be modified by mixing the material with the solvent using magnetic stirrer, mechanical stirrer or by shaking occasionally using hand.
   • The extract is filtered and fresh solvent is added after 8-12hrs.
   • The process is repeated until all the desired solutes are extracted.
   • It is used for the tougher plant parts or those that consist poorly soluble substances.

4. • Infusion:

   • The plant material is macerated in boiling water for a short period of time.
   • In this process, chemical compounds or flavours from plant material are siphoned in a solvent as water, oil or alcohol, by permitting the material to stay suspended in the solvent over a period of time.

5.  Decoction:

   • The crude plant material is boiled in a specified volume of water for a given time frame.
   • It is then cooled and strained or filtered.
   • Decoction time varies depending on the uniformity of the parts to extract.
   • It is suitable for extracting water soluble and heat stable constituents.
   • Decoctions are prepared for fresh purpose and shouldn’t be stored for more than 24 hours.
   • Decoction may be used to prepare herbal teas, leaf teas, coffees, tinctures and similar solutions.

Extraction with boiling solvents (Refluxion):

• The plant material is treated with boiling solvent and hence is a hot extraction process.
  The solvent vapor is recycled by a condenser fitted on top of the container, preferentially a round bottomed flask.

1.  Hot Continuous Extraction (Soxhlet):
   • It is named after ‘Franz Ritter von Soxhlet’, a German agricultural chemist.
   • It is the convenient method for the continuous extraction of a solid by a hot solvent.
   • In this process, the transfer of partially soluble components of a solid to the liquid phase takes place using a Soxhlet extractor.
   • The finely ground crude plant material is placed in a thimble, which is made of strong filter paper that allows liquid to pass through and is placed inside the Soxhlet apparatus.
   • The apparatus is then fitted to a round bottomed (RB) flask containing the solvent and to a reflex condenser.
   • The solvent in the RB flask is boiled mildly.
   • The vapor passes up through the side tube, gets condensed by the condenser and falls into the thimble containing the material and slowly fills the Soxhlet.
   • When the solvent reaches the top, it extracts over into the flask, removing portion of the substance which it has siphoned and the process repeats.
   • After extraction, solvent is eliminated by means of a rotary evaporator, resulting extracted compound.
   • The non-soluble portion of the extracted solid stays in thimble and is usually relinquished.
   • Advantages:
   • Large amount of drugs can be extracted with much smaller quantity of solvent.
   • More economical and feasible on medium or large scale.
2. • Steam Distillation:
   • It is used for the separation of essential oil from crude plant material.
   • Simple vaporization is gained by passing steam directly through the material.
   • The steam vaporizes the plant material’s volatile compounds which finally pass through a condensation and collection process.
   • Since water and oil do not mix in collection process, the essential oil floats on top of the water and it is extracted off.
3. • Hydro Distillation:
   • It is used instead of steam distillation.
   • The plant material is soaked in water and boiled by use of heating mantle.
   • Due to the impact of hot water, the essential oil is freed from the oil glands in the plant tissues and passed along with the steam.
   • In a typical glass apparatus known as Clevenger apparatus, the steam oil mixture is condensed and oil is separated from water and the condensed water is recycled.
4. • Enfluerage:
   • It is used for the extraction of delicate fragrances as that of some flowers.
   • The flower petals are spread over a layer of refined fat which picks up the odour of the flowers.
   • The saturated fat is handled with a solvent, usually alcohol in which the fragrant components are soluble.
   • The residual fat dissolved in alcohol may be eliminated by cooling the alcohol extract to 20oC, when fat separates out.
   • The volatile components are then retrieved from alcohol by concentrating the solution at reduced pressure in a rotavapor.

• Supercritical Fluid Extraction (SFE):

• Critical point represents the conditions above which distinct liquid and gas phases do not exist, but a homogenous supercritical fluid state exists.
• Supercritical fluid is obtained by heating above the critical temperature and compressing above the critical pressure and has the properties of a liquid as well as that of a gas.
• Alternative method with reduced use of organic solvents and increased sample throughput.
• Most commonly used supercritical fluid is CO2 .
• The lower viscosities and higher diffusion rates of supercritical fluids compared to liquids amplifies the extraction process.
• It is non inflammable, chemically inert, odour free, easy for disposal and economical with high purity and also can be recycled.
• Other gases such as ethylene, ethane, propylene, propane and nitrous oxide can also be used.
• High pressure carbon dioxide can be applied in place of various traditional organic solvents and steam distillation.
• Above 1100 psi and 31.70C, carbon dioxide reaches in “supercritical region.”
• Under these conditions, it has the solvating power of a liquid and the diffusion property of a gas.
• Carbon dioxide, being a supercritical fluid is a superior solvent for the extraction of a wide variety of natural products.
• This technique has been in use in industry for decaffeination of coffee and the removal of nicotine from tobacco.
• It is a more efficient method for the extraction of taxol and baccatin from the Yew tree.
• The major advantage is the implementation of mild conditions, which avoid the risk of thermal degradation compared to distillation and solvent extraction.

• Ultrasonic Extraction:

• It involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz.
• It increases the porosity of cell walls and produces cavitation.
• Ultrasound aided extraction can be used with mixtures of immiscible solvents such as hexane with methanol/water.
• In cases where the process creates heat, the extraction container is placed in ice bath to prevent heat labile compounds from decomposition.

• Microwave Assisted Extraction:

• Microwaves are electromagnetic radiations with the frequency range 0.3 to 300 GHz.
• It is made up of two oscillating perpendicular fields: electrical field and magnetic field.
• They can be used as information carriers or as energy vectors.
• Ionic conduction and dipole rotation in both the solvent and the sample which efficiently changes microwave energy to thermal energy.
• If the solvent selected has a high dielectric constant it strongly absorbs the microwave energy.
• However, in some cases, only the sample matrix may be heated, so that the solutes are freed in a cold solvent to avoid the degradation of thermolabile compounds.
• Microwave energy is applied to the sample suspended in solvent, with short intervals of cooling time.
• It’s merits include e.g., shorter extraction time, less solvent, higher extraction rate and lower cost.

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What is Bronchitis?

• Bronchitis is defined as persistent cough with sputum production for atleast 3 month in 2 consecutive years

There are three types of bronchitis:

1. simple chronic bronchitis: it Is a kind of bronchitis in which cough is present with no physiologic evidence of airflow obstruction.
2. chronic asthmatic bronchitis: it is kind of bronchitis in which hyper reactive airways is present with intermittent bronchospasm and wheezing
3. obstructive chronic bronchitis: it is a kind of bronchitis in which there is development of chronic airflow obstruction_ emphysema in a heavy smokers

What are the causes of Bronchitis?

• Chronic irritation by inhaled substances
• Microbial infection
• Middle age
• Cigarette smoking
• Other pollutants like sulfur dioxide, nitrogen dioxide,
• Hyper secretion of mucus in large airways- hallmark of chronic bronchitis
• Hypertrophy of the submucosal glands of trachea and bronchioles
• Marked increased in goblet cells of small airways

What is the Pathophysiology of Bronchitis?

• Hypermeia, swelling of mucus membrane
• Increased number of goblet cells
• Bronchial wall become thickened, bronchial lumen is narrowed
• Mucus may plug airwayAlveoli adjacent to bronchioles may damaged and fibrosed
• Respiratory infection
• Bronchitis

What are the clinical features of Bronchitis?

• Persistent productive cough with copious sputum
• Dyspnea on exertion
• Hypercapnia, hypoxemia, and mild cyanosis
• Cor-pulmonale with cardiac failure
• Chest pressure, headache, shortness of breath, sleeping difficulty,or sore throat
• Fatigue or malaise
• death

What are the diagnostic evaluation for Bronchitis?

• history taking: take the history of the patient like personal history, history of smoking, history of bronchitis in family member, allergy history, history of infection, any past medical and surgical history

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Principle:

• Soil is the prime storage of the microorganisms producing antibiotics which are able to inhibit the growth of other microorganisms. Antibiotics have been implemented in one or other forms for centuries. The screening of wild isolates from the soil has yielded the broad majority of new antibiotics. Even if the purification of several hundred naturally produced antibiotics has been performed, only a few have been proved to be successful to be used in medical practice. Those which are presently of greatest use were derived from a comparatively small group of microorganisms belonging to the genera Penicillium, Streptomyces, Cephalosporium, Micomonospora and Bacillus. In this era, the continuous efforts to develop new antibiotics are the emerging trends.
• Even if soils from various parts of the world are continually screened in industrial laboratories in order to isolate new antibiotic-producing microorganisms, industrial microbiology is directing its efforts toward chemical modification of existing antibiotic substances. This is completed by adding or replacing chemical side chains, reorganizing intramolecular bonding, or producing mutant microbial strains able to excrete a more potent form of the antibiotic. The establishment of chemical congeners accounts for the overcoming of antibiotic resistance, reducing adverse side effects in the host and increasing the effective spectrum of a given antibiotic.
• I: We will use the crowded-plate technique for the isolation of antibiotic producing microorganisms from two soil samples, one of which is seeded with Streptomyces griseus to serve as a positive control.
• II: For the determination of anti-microbial spectrum of isolates, isolates manifesting antibiotic activity will be screened against several different microorganisms to establish their effectiveness.

I. Isolation of antibiotic producing microorganisms

Requirements:

1.  Soil Suspensions:
   • – 1:500 dilution of soil sample suspension (0.1 g of soil per 50 ml of tap water) to serve as an unknown
   • – 1:500 dilution of soil sample seeded with S. griseus (0.1 g of soil per 50 ml of tap water) to serve as a positive control.
2. Media:
   • Six 15-ml Trypticase soy agar deep tubes, and two Trypticase soy agar slants.
3. Equipment:
   – 500-ml beaker
   – test tubes
   – test tube rack
   – sterile Petri dishes
   – inoculating needle
   – hot plate
   – thermometer
   – 1-ml and 5-ml pipettes
   – mechanical pipetting device
   – magnifying hand lens.

Procedure for Isolation of antibiotic producing microorganisms

• Label two sets of three sterile Petri dishes with the types of soil samples being used and dilutions (1:1000, 1:2000, and 1:4000).
• Place six Trypticase soy agar deep tubes into a beaker of water and bring to 100°C on a hot plate. Once agar is liquefied, add cool water to the water bath. Cool to 45°C, checking the temperature with a thermometer.
• Prepare a serial dilution of the unknown and positive control 1:500 soil samples as follows:
  – Label three test tubes 1, 2, and 3. With a pipette, add 5 ml of tap water to each tube.
  – Shake the provided 1:500 soil sample thoroughly for 5 minutes to effect a uniform soil-water suspension.
  – Using a 5-ml pipette, transfer 5 ml from the 1:500 dilution to Tube 1 and mix. The final dilution is 1:1000.
  – Using another pipette, transfer 5 ml from Tube 1 to Tube 2 and mix. The final dilution is 1:2000.
  – Using another pipette, transfer 5 ml from Tube 2 to Tube 3 and mix. The final dilution is 1:4000.
  – Using separate 1-ml pipettes, transfer 1 ml of the 1:1000, 1:2000, and 1:4000 dilutions to their appropriately labelled Petri dishes.
  – Pour one tube of molten Trypticase soy agar, cooled to 45°C, into each plate and mix by gentle rotation.
  – Allow all plates to solidify.
• Incubate all plates in an inverted position for 2 to 4 days at 25°C.
• Examine all crowded-plate dilutions for colonies exhibiting zones of growth inhibition. Use a hand magnifying lens if necessary. Record in the Lab Report the number of colonies showing zones of inhibition.
• Aseptically isolate one colony showing a zone 34 of growth inhibition from each soil culture
  with an inoculating needle and streak onto
  Trypticase soy agar slants labelled with the soil sample from which the isolate was obtained

• Incubate the slants for 2 to 4 days at 25°C. These will serve as stock cultures of antibiotic-producing isolates to be used in Part B.

II. Determination of antimicrobial spectrum of isolates

Requirements:

1. Cultures:
   • – 24-hour Trypticase soy broth cultures of Escherichia coli, Staphylococcus aureus, Mycobacterium smegmatis, and Pseudomonas aeruginosa.
2. Media:
   Two Trypticase soy agar plates.
3. Equipment:
   – Bunsen burner
   – inoculating loop
   – glassware marking pencil.

Procedure  for determination of antimicrobial spectrum of isolates.

• Label the Trypticase soy agar plates with the soil sample source of the isolate.
• Using aseptic technique, make a single-line streak inoculation of each isolate on the surface of an agar plate so as to divide the plate in half
• Incubate the plates in an inverted position for 3 to 5 days at 25°C.
• Following incubation, on the bottom of each plate draw four lines perpendicular to the growth of the antibiotic-producing isolate
• Aseptically make a single-line streak inoculation of each of the four test cultures following the inoculation template on each plate. Start close to, but not touching, the growth of the antibiotic-producing isolate and streak toward the edge of the plate.
• Incubate the plates in an inverted position for 24 hours at 37°C.
• Examine all plates for inhibition of test organisms, and record your observations in the Lab Report.

Observations and Results interpretations:

• I: Isolation of Antibiotic-producing micro-organisms.
  -Number of colonies showing zone of inhibition in different serial dilutions were noted and were further cultured to obtain pure cultures.
• II: Determination of anti-microbial spectrum of isolates.
  • Draw a representation of the observed antibiotic activity against the test organisms.
  • Based on your observations, record in the chart the presence (+) or absence (−) of antibiotic activity against each of the test organisms and the spectrum of antimicrobial activity (broad or narrow).

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Principle:

• Members of the anaerobic genera Clostridium and Desulfotomaculum and the aerobic genus Bacillus are examples of organisms that have the capacity to exist either as metabolically active vegetative cells or as highly resistant, metabolically inactive cell types called spores. When environmental conditions become unfavorable for continuing vegetative cellular activities, particularly with the exhaustion of a nutritional carbon source, these cells have the capacity to undergo sporogenesis and give rise to a new intracellular structure called the endospore, which is surrounded by impervious layers called spore coats. As conditions continue to worsen, the endospore is released from the degenerating vegetative cell and becomes an independent cell called a free spore. Because of the chemical composition of spore layers, the spore is resistant to the damaging effects of excessive heat, freezing, radiation, desiccation, and chemical agents, as well as to the commonly employed microbiological stains. With the return of favorable environmental conditions, the free spore may revert to a metabolically active
  and less resistant vegetative cell through germination. It should be emphasized that sporogenesis and germination are not means of reproduction but merely mechanisms that ensure cell survival under all environmental conditions.

In practice, the spore stain uses two different stains and decolorizing agents:

1. Primary Stain (Malachite Green):

• Unlike most vegetative cell types that stain by common procedures, the free spore, because of its impervious coats, will not accept the primary stain easily. For further penetration, the application of heat is required. After the primary stain is applied and the smear is heated, both the vegetative cell and spore will appear green.

2. Decolorizing Agent (Water):

• Once the spore accepts the malachite green, it cannot be decolorized by tap water, which removes only the excess primary stain. The spore remains green. On the other hand, the stain does not demonstrate a strong affinity for vegetative cell components; the water removes it, and these cells will be colorless.

3. Counter stain (Safranin):

• This contrasting red stain is used as the second reagent to color the decolorized vegetative cells, which will absorb the counterstain and appear red. The spores retain the green of the primary stain.

Requirements

• i). Bacterial Culture: 48-72 hrs nutrient agar slant culture of Bacillus cereus and thioglycollate culture of Clostridium sporogenes.
• ii. Reagents: Malachite green and safranin.
• iii. Equipment: Microincinerator or Bunsen burnerhot plate, staining tray, inoculating loop, glass slides, bibulous paper, lens paper, and microscope.

Procedure for Spore staining:

1. Obtain two clean glass slides.
2. Make individual smears in the usual manner using aseptic technique.
3. Allow smear to air-dry, and heat fix in the usual manner.
4. Flood smears with malachite green and place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes. Note: Do not allow stain to evaporate; replenish stain as needed. Prevent the stain from boiling by adjusting the hot
   plate temperature.
5. Remove slides from hot plate, cool, and wash under running tap water.
6. Counterstain with safranin for 30 seconds.
7. Wash with tap water.
8. Blot dry with bibulous paper and examine under oil immersion.
9. In the chart provided in the Lab Report, complete the following:
   • Draw a representative microscopic field of each preparation.
   • Describe the location of the endospore within the vegetative cell as central, sub-terminal, or terminal on each preparation.
   • Indicate the color of the spore and vegetative cell on each preparation

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What is Puerperium?

• Puerperium is a duration in which the reproductive organs and all the system of the body revert to their normal condition.
• It is followed by the delivery of the placenta and ends approximately 6 weeks later.
• Puerperium can also be defined as a period following childbirth in which the body tissues, particularly the pelvic organs return back approximately to the pre-pregnants state both anatomically and physiologically.
• Puerperium or post partum period is term given for the first 6 weeks following the birth of an infant.
• Mothers experience several physiologic and psychological changes during this time.
• They are listed as follows:

• • Reverting of the reproductive organ to their pre-pregnant stage.
  • Initiation of Lactation.
  • Recovery of the mother from the physical and emotional experiences of parturition.
  • The foundations of the relationship between the infant and its parents are established.

A. Physiological changes in reproductive system:

I. Involution of the uterus:

• Involution is a term given to the changes that the reproductive organs, specifically the uterus, goes through after their childbirth to return to their pre pregnancy size and condition.
• Involution relies on three processes:
  • a. Contraction of muscle fibres
  • b. Catabolism
  • c. Regeneration of uterine epithelium

a. Contraction of muscle fibres:

• The involution starts immediately after placenta delivery when uterine muscle fibers contact around maternal blood vessels at the region where the placenta has been attached securely.
• As the muscle fibers that have been stretched for several months contract and gradually recover their original contour and size, the uterus decreases in size.

b. Catabolism:

• Although the total number of cells remains unchanged, catabolic changes in protein cytoplasm are experienced in the enlarged muscle cells of the uterus that cause a decrease in individual cell size.
• The catabolic process products are absorbed by the blood stream and are excreted as nitrogenous waste in urine.

c. Regeneration of uterine epithelium:

• Soon after childbirth, regeneration of the uterine line begins.
• With the placenta, the outer part of the endometrial layer is expelled within 2-3 days and the remaining deciduas are divided into two layers.
• The initial layer is superficial and is shed in lochia.
• The basal layer remains intact and is the new endometrium source.
• Endometrium regeneration, except at the site of placental attachment, occurs within 2-3 weeks.
• The placental site contracts rapidly providing an elevated surface measuring approximately 7.5cm and stays elevated even at 6 weeks, until it measures approximately1.5cm.
• Healing occurs more slowly at the placental site and takes about 6-7 weeks.
• The uterus is in the midline at the end of the third stage of labor, about 2cm below the umbilicus level.
• The uterus weighs about 1000gm at this time.
• Within 12 hrs, the fundus can rise to approximately 1 cm above the umbilicus. 
• The uterus is about the same size at 24 hours postparutm as it was at 20 weeks of gestation. Involution will develop rapidly over the next few days.
• The fundus descends every 24 hours by around 1-2 cm or 1 finger, so that it is in the pelvic cavity by the 8-10th day and can not be palpated abdominally.
• And there are individual variations linked to body size.
• The uterus, which weighs approximately 11 times its pregnancy weight in full, involutes approximately 500 gm per 1 week after birth and 300-350 gm per 2 weeks after birth.
• It weighs 60gm in 6 weeks.
• Increased levels of estrogen and progesterone are responsible for promoting massive uterine growth during pregnancy.
• Prenatal uterine development results from both hyperplasia, an increase in the number of muscle cells, and an enlargement of existing cells due to hypertrophy.
• The reduction of these hormones postnatally induces autolysis, the self-destruction of excess hyperthyroid tissue.
• The powerful frequency of myometrial contractions that regulate the flow of blood to the uterus stops, making it difficult.
• By palpating the uterus, its consistency can be measured. It ought to feel firm and round.

II. Cervix:

• The cervix is formless, flabby, and open enough to accommodate the entire hand immediately after birth.
• This makes it possible, if appropriate, to manually remove the placenta and to manually inspect the uterus.
• There can be minor tears and lacerations, and the cervix is sometimes edematous.
• Rapid healing happens and the cervix feels firm by the end of the first week.
• For the first 4-6 days postpartum, two fingers may still be inserted into the cervical os, but only the smallest curette can be introduced by the end of 2 weeks.
• The external cervical os never acquires its prepregnant appearance, it is no longer shaped like a circle, but appears as a jagged slit that is sometimes portrayed as a fish mouth.

III. Vagina:

• During birth, the vagina and the vagina introitus are substantially extended to enable the fetus to move.
• The vaginal walls appear edematous, smooth, soft and some minor lacerations may be present soon after childbirth.
• Vaginal mucosa becomes atrophic during the postpartum periods, and vaginal walls do not recover their thickness until ovarian estrogen production is reestablished.
• Due to ovarian activity, and thus the development of estrogen during lactation is not well known, breast feeding mothers are likely to experience vaginal dryness and may experience intercourse discomfort.
• Estrogen deficiency is also accountable for a reduced amount of vaginal lubrication.
• In the vaginal condition, the introitus remains permanently larger.
• The hymen is lacerated and is expressed by nodular tags.
• Adequate suturing has been done in well-healed vaginal tears.
• The vagina shrinks to a non-pregnant level, but it does not return to its pregnant size fully.

IV. Perineum:

• During the second stage of labor, as the fetal head applies pressure as it descends, the pelvic floor muscle stretches and thins considerably and rotates and then expands to be delivered.
•  And an intact perineum may be edematous, erythematous and painful after delivery.
•  Swelling and tenderness as a result of the birth of a baby are initially present.
•  Healing of an episiomoty is identical to any surgical incision.
• Healing should occur between 2-3 weeks.
• When episiotomy and perineal tears are done, a scar may be present.
• Pelvic floor supporting tissue that is torn or stretched during childbirth can take up to 6 months to recover tone.
•  Kegel exercises which helps enhance perineal muscles and promote healing are suggested after childbirth.
• Adequate suturing has been done with well episiotomies and perineal tears.

V. Ovaries:

• The resumption of the ovaries’ regular function is highly variable and is profoundly affected by the breastfeeding of infant.
• The woman who breastfeeds the baby has a longer amenorrhea and ovulation cycle than the mother who does not breastfeed will ovulate after 27 days of delivery.
• Most women have a menstrual cycle of 12 weeks, with a mean duration of 7-9 weeks for the first menstrual.

VI. Lochia:

• Lochia is vaginal discharge after child birth.
• The uterine body, cervix and vagina are the sources of the discharge.
• Blood leucocytes, decidua sheds, and organisms compose the lochia.
• Initially, the lochia is bright red, but after the first week the color fades and the flow usually clears entirely within 4 weeks of delivery.
•  As involution progresses, postchild birth uterine discharge undergoes sequential modifications.
• Lochia rubra: consist primarily of blood, sheds of fetal membranes and decidua, vernix caseosa, lanugo. It may consist few small blood clots.
• Lochia serosa: fewer RBC, more leucocytes, serum, mucus, and tissue debris. These are pink colored and are released over the next 5-9 days.
• Lochia alba: contains large number of deciduous cells, leukocytes, mucus, serum, epithelial cells and bacteria. The discharges are colored pale, creamy, brown and last 10-14 days. Any signs of discharged stained blood may continue to be seen for a further 2-3 weeks. The color of lochia indicates the placental site’s healing period.
• Odor and reaction:
  • It’s got a distinct unpleasant fishy scent.
  •  Its reaction is alkaline, tending to become acid at the end.
• Amount:
  • Estimating the quantity of lochia is difficult.
  •  Due to absorption in pads, sari, etc., the true amount may be concealed.
  • The weight of the pads can also be weighed and compared with the weight of the clean dry pad (1 g of weight equal to 1 ml) or based on the amount of stain on the perineal pad, providing a definition and an approximation in milliliters of 1 hour for lochia.
  • The average discharge level is calculated to be 250ml for the first 5-6 days.
  • Scanty: less than one 2 inch (5cm) stain in one hour on the peri pad= 10 ml
  • Light: stain on the perineal pad less than 4 inches (10cm) within 1 hour= 10ml to 25ml
  • Moderate: less than 6 inches (15cm) of stain within 1 hour on the perineal pad= 25-50ml.
  • Heavy: greater than 6 inches or heavy saturated pad= 50-80 ml within 1 hour.
• Normal charateristics of lochia:
  • Lochia rubra is 1-4 days in length. Bloody,small clots.
  • Lochia serosa is 5-9 days long. Decreased amount, serosanguneous, pink or brown.
  • For 10-15 days, Lochia aalba lasts. Creamy, yellowish color, decreasing amounts.
• Clinical importance:
  • Useful knowledge about the irregular puerpural condition is provided by the character of the lochia discharge.
  • – Severe lochia suggests infection when offensive.
  • – if scanty, denotes infection or lochiometra
  • – If persistence of red color further than normal limit suggests subinvolution or retained bits of conception.
  • – Local genital lesion is suggested when it lasts past 3 weeks.

B. Physiological changes in other systems of body:

1. Vital signs:

• 1. Pulse:
  • The pulse rate is likely to be raised for a few hours after normal delivery, calming down to normal during the second day.
  • The pulse rate, however, can also increase with pain or excitement afterwards.Any tachycardia (pulses > 110 or more bpm) may be suggestive of severe bleeding or the development of puerperal infection.
• 2. Temperature:
  • As an usual physiological reaction, the temperature may be labile within the first few days following delivery.
  • Temperatures within the first 24 hours should not be above 37.2^o C.
  • After delivery, there could be a small reactionary increase of 0.5^o F, but within 12 hours it comes down to normal.
  • Due to breast engorgement, which does not last for more than 24 hours, there might be a minor temperature increase on the 3rd day.
  • Puerperal pyrexia results from genital or urinary tract infection, breaches or inflammation within the venous system.
• 3. Blood pressure:
  • Because of an increased venous return, there may be a slight rise in the blood pressure.
  • Blood pressure differs with position and in order to gain accurate results, it should be measured with the mother in the same position each time.
  • A rise from the baseline indicates hypertension caused by pregnancy, a decrease may indicate dehydration or hypovolemia due to excessive bleeding.
  • Conduct a quick initial test.

2. Respiration:

• It is necessary to maintain a normal respiration rate of 16-20 per minute.

3. Gastrointestinal System:

• i. Appetite:
  • Shortly after birth, the mother is normally hungry and can handle a light diet.
  •  New mother is normally hungry due to the extreme energy lost in labor.
  • Besides that she is generally thirsty because of fluid loss during labor, in the lochia, diuresis and prespiration.

•  
• ii. Bowel evacuation:
  • For 2-3 days after childbirth, a bowel evacuation may not occur.
  • The decreased muscle tone in the intestine during childbirth and the immediate puerperium, prelabour diarrhea, lack of food or dehydration may explain this delay.
  • The mother often observes discomfort during the bowel movement due to lack of perineal muscles, reflex pain in the perineal region, slight intestinal paresis are factors contributing for constipation.
  • When the bowel tone returns, normal bowel patterns should be reestablished.
  • The strain and pressure on the lower bowel triggers the extrusion of internal hemorrhoids during delivery.
  • They decrease in size after delivery and can be manually re-inserted into the rectum.
  •  Hemorrhoids present during pregnancy often shrink and occasionally surgical reduction.
  • The rate at which the intestine is regulated depends on everyday life, food and fluids, exercise.

4. Neurologic System:

• Induced neurological pain in pregnancy disappears after birth.
• Removal of physiologic edema through the diuresis that accompanies childbirth relieves carpal tunnel syndrome by inducing compression of median nerve.

5. Integumentary System:

• Chloasma of pregnancy typically disappears at the end of pregnancy.
• After childbirth, areola and linea nigra hyperpigmentation does not regress entirely.
• These areas may have permanent darker pigmentation for some women.
• Breast, abdomen and thigh striae gravidarum (stretch marks) can fade (silvery color in light skinned women) but typically don’t disappear.
• Hair and nail development can return to pre-pregnant patterns in a few months.

6. Respiratory System:

• The diaphragm descends to its usual location after delivery, which decreases abdominal pressure, allowing for improved lung expansion and ventilation, but the respiratory rate does not change significantly.

7. Urinary System:

i. Physical changes:

• When the fetal head moves under the uterus, the urethra, bladder and tissue around the urinary meatus may become edematous and traumatized during childbirth.
•  This also results in reduced fluid pressure sensitivity, even though the bladder is distended.
•  Owing to the diuresis that accompanies childbirth, the bladder fills easily.
•  As a result, the mother is at risk for over distention of the bladder, incomplete emptying of the bladder.
•  Body water in the extra vascular spaces and excess plasma volume from pregnancy are quickly removed.
• Yet diuresis and polyurea occur up to 3 liters/day on the second postpartum day.
•  The urine passes for a few days and returns to the usual voiding pattern after one week.
•  Bladder boosts its ability, filling up to 1000 or 1500 ml of urine without pain.
•  Regional or general anesthesia can inhibit normal function temporarily, diminishing the bladder urinary sensation.
•  The woman at risk for haemorrhage from a poorly contracted uterus is followed by urinary retention.
•  Stasis also predispose to urinary tract infection.
• Weight Loss:
  • During childbirth, about 5.5 kg (12 pounds) of weight is lost.
  • This involves the weight lost during the birth of the fetus, placenta and aminotic fluid and blood.
  • During the first 2 weeks following childbirth, an additional 2-4 kg is lost.
  •  This includes the weight lost during the first few post partum days by diuresis and diaphoresis.
• Fluid loss:
  • Total fluid loss for the first week of at least 2 liters and for the next 5 weeks of an additional 1.5 liters.
  • The loss amount depends on the amount returned during the prenatal and natal phases.

8. Musculo-Skeletal System

• Abdominal muscles: The uterine ligaments remain loose and relaxed, with less tone in the abdominal muscles, resulting in the abdomen becoming flexible and flabby.
• During the first days after birth, as the woman stands up, her belly protrudes and gives her a pregnant look.
• The abdominal wall is relaxed during the first 2 weeks after birth.
• It takes about 6 weeks for the abdominal wall to return to its state of nearly non-pregnancy.
• The restoration of muscle tone relies on the previous tone, proper exercise, and the amount of adipose tissue.
• The abdominal wall muscle distinguishes a disorder called diastasis recti abdominis sometimes with or without overdistension due to a large fetus.
• Joints: The pelvic joint, especially the symphysis pubis, can separate slightly during labor under the influence of relaxation, causing pain and discomfort, stabilizing by 6-8 weeks.

9. Cardiovascular System:

• Change in blood volume: Changes in blood volume after birth depend on many factors, such as loss of blood during childbirth and mobilization and excretion of extravascular water (physiologic edema).
• Cardiac output:
  • Due to a rise in stroke volume, cardiac output tends to increase for at least the first 48 hours postpartum.
  • This increased volume of stroke is caused by the return of blood to the systemic venous circulation of the mother, resulting from a rapid reduction in the flow of uterine blood and extravascular fluid mobilization.
  •  By 6 weeks postpartum, cardiac output generally returns to normal.
  •  The heart rate and blood pressure return within a couple of days to non-pregnant levels.
  •  After delivery, body tries to compensate for increase central venous load, slowing the heart rate, to regulate cardiac output and avoid systemic overload and hypertension.
  • Hemorrhage, inflammation, thrombosis, anxiety, discomfort or excitement at delivery may be demonstrated by a rise in pulse rate.
  • In response to anesthesia, blood pressure may decrease in the early recovery period; orthostatic hypotension may occur due to fluid changes and reduced intra-abdominal pressure.
  •  It returns to normal within a few days after delivery, unless complications such as hypertension caused by pregnancy arise in women.
• Blood Components:
  • A greater decrease in plasma volume than in the amount of blood cells occurs within the first 72 hours after childbirth.
  • Haematocrit could rise in the first 3-7 days, slowly return to normal levels by 4-5 weeks as old cells die out and fewer new ones form.
• WBC count:
  • During first 10-12 days after child birth ,value between 20,000 and 25,000/mm³ are common. It falls to normal in 4-7 days.
  • Persistent elevation implies infection.
  • The large increase in WBCs is caused by neutrophils that increase in response to inflammation, pain and stress to protect against invading species.
• Coagulation factors:
  • During pregnancy, clotting factors and fibrinogen are typically increased and remain elevated in the immediate puerperium; during healing, platelet, fibrin and fibrinogen levels are elevated. Their function is to protect against bleeding.
  • This hypercoaguable condition causes an increased risk of thormboembolism when combined with vessel damage and immobility, in particular after cersarean birth.

10. Endocrine System

• The levels of estrogen and progesterone levels drop remarkably after expulsion of the placenta and reach their lowest levels 1 week postpartum.
• Reduced estrogen levels are related with breast engorgement and with the diuresis.
• In non-lactating women, estrogen begin to rise by 2 weeks after birth.
• Human chorionic gonadotropin (hCG) disappears from maternal circulation in 14 days.
• Oxytocin continues to acts upon the uterine muscle fibres maintaining their contraction, reducing the placental site and preventing haemorrhage.
• In women who choose to breast feed babies, the suckling of the infant stimulates further secretion of ocytocin and this aids the continuing involution of the uterus and expulsion of milk.
• Prolacting levels remain elevated in the sixth week after birth in women who breastfeed.
• The level of serum prolaction is affected by the frequency of breastfeeding, the length of each feeding, and the degree to which additional feeding is used.
• Individual variations in the intensity of the sucking stimulus of an infant probably also influence the levels of prolactin.
•  In woman who breast feed, the levels of prolactin remain high and the resumption of follicle stimIn women who are breast-feeding, prolactin levels remain high and the resumption of ovary follicle stimulation is suppressed.
• Prolactin levels decrease after birth in non-lactating women and enter the pregnant range by the two to third postpartum week; this enables the follicle stimulating hormone secreted by the anterior pituitary gland to act on the ovary, contributing to the restoration of normal patterns of development of estrogen and progesterone, follicle formation, ovulation, and menstruation.

11. Menstruation and Ovulation

• The occurrence of the first menstrual period following delivery is very variable and depends on lactation.
• If the woman If the woman does not breastfeed her infant, menstruation returns in around 40 percent by the 6th week after childbirth and in 80 percent of cases by the 12th week.does not breastfeed her baby, the menstruation returns by 6^th week following delivery in about 40% and by 12^th week in 80% of cases.
• Contractive protection for women who are primarily breastfeeding is roughly 98 percent up to 6 months postpartum.
•  Breastfeeding postpones the return of both menstruation and ovulation.
•  The duration of the delay depends on the duration of lactation and frequency of breastfeeding.
• Increased frequency, length of suckling is linked with high prolactin level, prolonged ovarian suppression and lactational amenorrhoea.

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• Biodiversity is defined as the variations among living organisms from all possible sources. It includes the variability within or between the species and within or between ecosystems.
• According to the definition of the 1992 UN conference on Environment and Development (UNCED) Convention, biodiversity includes all of its manifestations. Therefore, along with terrestrial biodiversity, it also covers marine as well as other aquatic biodiversity. As such biodiversity means the richness and variety of living things in the world as a whole or in any location within it.

What are the elements of biodiversity?

• Major elements of biodiversity comprise of- Ecosystem diversity, Species diversity, and Genetic diversity.
  

  1. Ecosystem diversity

• An ecosystem is made of a dynamic system of plant, animal, and microorganism groups and their non-living environment altogether interact as functional unit.
• Non-living components cover sunlight, air, water, minerals and nutrients.
• Ecosystem can be small and short-lived, for example, water filled tree holes or rotting logs on a forest floor or large and long-lived like forests or lakes. Thus, ecosystem commonly exist within ecosystems.
• Ecosystem diversity refers to the variation and rate of occurrence of distinct ecosystems including the variety of habitats, biotic communities and their change in structure and composition over time and ecological processes in the biosphere.
  

  2. Species diversity

• Species is defined as a population of organisms whose members are able to interbreed freely under natural conditions.
• A species represents a group of organisms which has evolved definite inheritable features and occupies a unique geographical area.
• Species usually do not freely interbreed with other species (Wilson,1992).
• Species diversity is used to describe the frequency and variety of species (wild or domesticated) within a geographical area.
• The total number of species in the globe has been estimated to range from 5-30 million (Wilson,1988), out of which approximately 1.7 million living species of all kinds of organisms have been described to date (WCMC,1992).
• The World Conservation and Monitoring Center suggests that there are many different ways to describe species diversity:
  • Species richness is the total number of species within a geographical area.
  • It is expressed as an enumeration of the species occurring within a particular sample area, and is one often used to measure species diversity.
  • Measures of species richness are the basis for the observation that diversity increases with decreasing latitude on Earth, for example, tropical areas are richer in species than temperate areas.
  • Species evenness is also used to measure species diversity which is expressed as relationship of species to each other.
  • This includes relative abundance of species in different categories.
  • It is also known as taxonomic diversity. For example, an island with two species of birds and one species of lizard has greater taxonomic diversity than an island with three species of birds but no lizards (Raven,1992).
  • Species dominance is expressed as the most abundant species as dominant (Botkin and keller,1995).

3. Genetic diversity

• Genes are the principal units of heredity which are passed from an organism to its offspring.
• These are composed of nucleic acids and are located along an organism’s chromosomes, in the plasmids of bacteria and other extra-chromosomal forms as well.
• Genes, either individually or in groups contribute different credits to an organism such as its physical appearance (black eyes or dark hair), its ability to resist certain pests, or survive drought
• Genetic diversity refers to contrast of genes and/or genomes within living organisms, that is, the genetic differences among populations of a single species and among individuals within a population.
• In other word this covers distinct populations of the same species such as hundreds of traditional rice varieties in Nepal.
• According to Raven (1992), it is also expressed as genetic variation within a population, such as genetic variation is very high among Indian rhinos, and very low among Cheetahs.
• Nature’s wild species contain valuable genetic information.
• If a species is to survive, it needs some genetic diversity. But an inbred population loses diversity, and becomes vulnerable to pests and infectious diseases which may endanger the whole population.
• Using ‘DNA fingerprinting’, molecular biologists can detect inbred population which may be moving forward for extinction.
• In the agriculture industry, monoculture crops, artificial insemination and embryo cloning technique lead to narrow, inbred population. In biomedicine too, genes from such species as fungi, lichens, marine organisms and higher plants have been used to produce antibiotics, anti-cancer agents, hormones, muscles relaxants, cardiac and respiratory stimulants.
• Modern biotechnology is generating recombinant DNA vaccines and pharmaceuticals, gene probes for inherited disease and forensic analysis, and genetically engineered organisms for mining, energy, chemical production and treatment of waste products.
• Therefore, genetic heritage of the earth once preserved can be read, appreciated and perhaps even reactivated by future generations.
  

  References:

• Chaudhary Ram P., M.Sc., Ph.D., D.Sc., F.N.R.S., Professor of Botany, TU, Kirtipur, Ktm, Nepal, Biodiversity in Nepal,1998.
• www.biologicaldiversity/org
• www.yourarticlelibrary.com

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