Nanoparticle

 A nanoparticle (or nanopowder or nanocluster or nanocrystal) is a microscopic particle with at least one dimension less than 100 nm.

Nanoparticle research is currently an area of intense scientific research, due to a wide variety of potential applications in biomedical, optical, and electronic fields.

Nanoparticles are of great scientific interest as they are effectively a bridge between bulk materials and atomic or molecular structures.

A bulk material should have constant physical properties regardless of its size, but at the nano-scale this is often not the case.

Size-dependent properties are observed such as quantum confinement in semiconductor particles, surface plasmon resonance in some metal particles and superparamagnetism in magnetic materials.

The properties of materials change as their size approaches the nanoscale and as the percentage of atoms at the surface of a material becomes significant.

For bulk materials larger than one micrometre the percentage of atoms at the surface is minuscule relative to the total number of atoms of the material.

The interesting and sometimes unexpected properties of nanoparticles are not partly due to the aspects of the surface of the material dominating the properties in lieu of the bulk properties.

Nanoparticles exhibit a number of special properties relative to bulk material.

For example, the bending of bulk copper (wire, ribbon, etc.) occurs with movement of copper atoms/clusters at about the 50 nm scale.

Copper nanoparticles smaller than 50 nm are considered super hard materials that do not exhibit the same malleability and ductility as bulk copper.

The change in properties is not always desirable.

Ferroelectric materials smaller than 10 nm can switch their magnetisation direction using room temperature thermal energy, thus making them useless for memory storage.

Suspensions of nanoparticles are possible because the interaction of the particle surface with the solvent is strong enough to overcome differences in density, which usually result in a material either sinking or floating in a liquid.

Nanoparticles often have unexpected visible properties because they are small enough to confine their electrons and produce quantum effects.

For example, gold nanoparticles appear deep red to black in solution.

Nanoparticles have a very high surface area to volume ratio.

This provides a tremendous driving force for diffusion, especially at elevated temperatures.

Sintering can take place at lower temperatures, over shorter time scales than for larger particles.

This theoretically does not affect the density of the final product, though flow difficulties and the tendency of nanoparticles to agglomerate complicates matters.

The large surface area to volume ratio also reduces the incipient melting temperature of nanoparticles.

Classification of Infections

Primary Infection- initial infection with a parasite in the host

Reinfection- subsequent infection by the same parasite

Secondary Infection- a new parasite infecting an IC host

Focal infection- infection at a localized site 

Cross infection- new infection est. From another host in a pt already suffering from ds

Nosocomial infections- HIC

Iatrogenic infection- through any therapeutic procedure

Causing an Infection

The endogenous – disease originates within the body.  Ex: metabolic disorders, congenital abnormalities, tumors.

The exogenous – disease originates outside the body.  Ex: chemical agents, electrical shock, trauma.

Nosocomial – acquired by an individual in a health care facility (workers to a patient). Many are antibiotic-resistant, life-threatening.

Opportunistic – occur when the body’s defenses are weak. Ex: pneumonia w/AIDs.

Source of Infection

• Humans from pt or carrier
• A healthy carrier
• Convalescent carrier
• Temporary carrier
• Contact carrier
• Paradoxical carrier
• Animals
• Insects vectors
• Mechanical vector
• Biological vector
• Soil and water 
• Food

Basic steps in Prevention of Infection

There are possible treatment and prevention to stop the infection cycle. This is through adequate hygiene, sanitary environment maintenance, and health education. 

Antimicrobial agents In Infection

• Anti-infective drugs such as antibiotics, antiviral, antifungal, and antitubercular drugs suppress infection.
• It can be administered by mouth, topically, or intravenously depending on the infection extent and severity.
• Sometimes, if drug resistance is known, multiple drugs are used to stop drug resistance and increase drug effectiveness. 
• Antibiotics only work for bacterial infection and have no effect on viral ones. 

History of infection control

1843 Oliver Wendell Holmes: contagious disease or communicable disease can be spread directly or indirectly from one person to another through contaminated hands. Ignaz Philipp Semmelweis observed a high mortality rate from MDs going from morgue to patients’ bedside without washing hands 1864 Joseph Lister: developed a surgical aseptic technique to prevent wound contamination.

Infection can be:

• Generalized or systemic (throughout the body)
• Localized (affecting one part of the body)

Signs and symptoms of infection:

• Systemic: headaches, fever, fatigue, vomiting, diarrhea, increased pulse and respiration
• Localized: redness, swelling, painful, warm to the touch

Chain of infection or Model of infectious disease transmission

Six elements must be present for an infection to develop

1.     The infectious agent 

2. Reservoir host

3. Portal of exit from the host

4.  Route of transmission

5.  Port of entry

6.  Susceptible host

1. Infectious agent: a pathogen must be present
2. Reservoir host: the pathogen must have a place to live and grow – the human body, contaminated water or food, animals, insects, birds, dead or decaying organic material. Humans who can transmit infection but how no signs of the disease are called carriers. A person may be unaware they are a carrier. 
3. Portal of exit: the pathogen must be able to escape from the reservoir host where it has been growing. Examples of portals of exit are blood, urine, feces, breaks in the skin, wound drainage, and body secretions like saliva, mucus, and reproductive fluids.
4. Route of transmission: When the pathogen leaves the reservoir host through the portal of exit, it must have a way of being transmitted to a new host. Examples of routes of transmission are air, food, insects, and direct contact with an infected person
5. Portal of entry: The pathogen must have a way of entering the new host. Common ports of entry are the mouth, nostrils, and breaks in the skin
6. Susceptible host: An individual who has a large number of pathogens invading the body or does not have adequate resistance to the invading pathogen will get the infectious disease

Breaking the chain of infection

Breaking at least one link stops the spread of infectious disease

1. The infectious agent - early recognition of signs of infection, Rapid, accurate identification of organisms 
2. Reservoir host - Medical asepsis, Standard precautions, Good employee health, Environmental sanitation, Disinfectant/sterilization
3. Portal of exit from the host, Medical asepsis, Personal protective equipment, handwashing, Control of excretions and secretions, Trash and waste disposal, Standard precautions 
4. Route of transmission - Standard precautions, Handwashing, Sterilization, Medical asepsis, Airflow control, Food handling, Transmission-based precautions
5. Portal of entry - Wound care, Catheter care, Medical asepsis, Standard precautions
6. Susceptible Host - Treating underlying diseases, Recognizing high-risk patients

Virulence and Pathogenicity

Pathogenicity: the capacity of microbes to cause disease

Virulence: the degree of pathogenicity of a specific microbe

Based on:

• Invasive qualities
• Toxic qualities
• Presence of pile or fimbriae for adhesion
• Ability to avoid host defenses (mutate)

Virulence Factors and Toxins

• Enzymatic Virulence Factors Examples: Coagulase (Staphylococcus aureus), Streptokinase (Streptococcus pyogenes), Hyaluronidase (Many pathogens), Collagenase (Many pathogens), Leukocidin (Many pathogens), Hemolysin  (Many pathogens)
• Adhesion Factors Examples: Protein A (Staphylococcus aureus), Protein M (Streptococcus pyogenes)
• Virulence factors help bacteria to, invade the host,  cause disease, and evade host defenses. 
• The following are types of virulence factors: 
• Adherence Factors: Many pathogenic bacteria colonize mucosal sites by using pili (fimbriae) to adhere to cells.
• Invasion Factors: Surface components that allow the bacterium to invade host cells can be encoded on plasmids, but more often are on the chromosome.
• Capsules: Many bacteria are surrounded by capsules that protect them from opsonization and phagocytosis
• Exotoxins - A type of bacterial toxin with the following properties: May be produced by either gram-positive or gram-negative bacteria, The action of the exotoxin does not necessarily require the presence of the bacteria in the host and Most exotoxins are peptide or protein 
• Most exotoxins are heat sensitive (exception: enterotoxin of Staphylococcus aureus)
• Exotoxins include several types of protein toxins and enzymes produced and/or secreted from pathogenic bacteria. Major categories include cytotoxins, neurotoxins, and enterotoxins.

Classes of exotoxins: Neurotoxic, cytotoxic, or enterotoxic exotoxins

• Neurotoxins: Interfere with proper synaptic transmissions in neurons
• Cytotoxins: Inhibit specific cellular activities, such as protein synthesis 
• Enterotoxins: Interfere with water reabsorption in the large intestine; irritate the lining of the gastrointestinal tract

Endotoxins

• A type of bacterial toxin having the following properties:
• Produced only by gram-negative bacteria 
• Endotoxins are a component of the gram-negative cell wall 
• The action of endotoxin requires the presence of the bacteria in the host. The endotoxin may be released from the cell wall as the cells die and disintegrate
• Endotoxin is composed of Lipid A: Part of the lipopolysaccharide layer 
• Mode of action: Irritation/inflammation of epithelium, GI irritation, capillary/blood vessel inflammation, hemorrhaging

• Endotoxins: The lipopolysaccharide endotoxins on Gram-negative bacteria cause fever, changes in blood pressure, inflammation, lethal shock, and many other toxic events.

Bacterial Identification Techniques

Accurate and definitive bacterial identification is essential for correct disease diagnosis, treatment of infection, and trace-back of disease outbreaks associated with microbial infections. Bacterial identification is used in a wide variety of applications including microbial forensics, criminal investigations, bio-terrorism threats, and environmental studies.

Techniques include:

• Conventional methods – Biochemical tests
• Antibody-based methods
• Nucleic acid-based methods – PCR, Southern blot, nucleic acid hybridization, RFLP, DNA fingerprinting
• Automated microbial identification methods
• DNA barcoding 
• Other methods – Phage typing, Flow cytometry, SDS

Bacterial Identification by Biochemical tests

Primary test

• Morphology
• Gram’s staining/Acid fastness
• Spores
• Motility
• The ability to grow in the air
• Ability to grow in the anaerobic conditions
• Catalase test
• Oxidase test
• Oxidation –Fermentation test

Secondary Tests

• Acetylmethylcarbinol production (VP) test
• Bile solubility test
• CAMP test
• Carbohydrate breakdown test
• Carbon sources test
• Chitinolytic test, Coagulase test
• Decarboxylase test
• Denitrification test
• Deoxyribonuclease test
• Gelatin hydrolysis test
• Haemolysin production test
• Hippurate hydrlysis test
• Hydrogen sulfide production test
• Indole test, Malonate test
• Methyl red (MR) test, O/129 sensitivity test
• ONPG test, Urease activity test
• Tween 20/80 hydrolysis test

Plasmid Isolation Using Alkaline Lysis

Plasmid Isolation Protocol

1. 5 ml LB medium containing proper antibiotics were inoculated with a single bacterial colony. The tube was incubated at 37 ˚C overnight with vigorous shaking at 360 rpm.
2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
3. Discard the supernatant.
4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipet up and down or vortex as necessary to fully resuspend the bacteria.
5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex.
6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle inversion. Do not vortex.
7. Centrifuge at 15,500 x g for 30 minutes at 4C.
8. Recover resulting supernatant.
9. Add 2.5 volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by repeated gentle inversion. Do not vortex.
10. Centrifuge at 15,500 x g for 30 minutes at 4C.
11. Removal of resulting supernatant. The pellet is plasmid DNA.
12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.
13. Add ddH2O or TE to dissolve the pellet. After the addition of 2ul RNase A (10mg/ml), the mixture was incubated for 20 minutes at room temperature to remove RNA. 

Note:

• Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this stage.
• Discard the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Q-tip.
• Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals (primarily magnesium and calcium). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and prevent the buffer from bursting the cells. 
• Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline solution gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for five minutes (but no longer, or your DNA will be irreversibly denatured). 
• Three things happen during this stage: 

1. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate) is a detergent found in many common items such as soap, shampoo, and toothpaste.
2. NaOH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA.
3. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated. Plasmid DNA is circular and remains topologically constrained.

• Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc), which does three things:

1. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single-stranded DNA (ssDNA).
2. The ssDNA is precipitated since large ssDNA molecules are insoluble in high salt.
3. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will allow for the easy removal of the SDS from your plasmid DNA. 

Now that you've made it easy to separate many of the contaminants, centrifuge to remove cell debris, KDS, and cellular ssDNA. Your plasmid DNA is in the supernatant, while all of the garbage is in the pellet.

• Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin this down. DNA is negatively charged, so adding a salt mask the charges and allows DNA to precipitate. This will place your DNA in the pellet.
• Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.
• Resuspend your now clean DNA pellet in the buffer (often Tris) and EDTA plus RNases to cleave any remaining RNA. Your DNA is now back in solution. 

DNA of this purity is good for a number of uses, such as in vitro transcription or translation or cutting with some enzymes. If you are sequencing or transforming this DNA into mammalian cells, you'll want to use additional purification techniques such as phenol extraction, Qiagen column purification, or silica-based purification. 

Hemagglutination (HA) Assay Protocol

The hemagglutination assay is a method for titering influenza viruses based on their ability to attach to

molecules present on the surface of red blood cells. A viral suspension may agglutinate the red blood

cells, thus preventing them from settling out of suspension. By serially diluting a virus in a 96-well plate and adding a consistent amount of red blood cells, an estimation of the amount of virus present can be made. 

Equipment and Materials Required

• Certified Biological Safety Cabinet
• Tabletop centrifuge with appropriate fittings
• Inverted microscope (optional)
• 15 ml conical tubes
• Disposable pipettes – 1 ml, 5 ml, 10 ml
• Micropipette and sterile disposable aerosol resistant tips – 160 µl
• PBS
• Turkey red blood cells in Alsevers solution purchased from a supplier such as Lampire Biological Products
• round-bottomed 96-well dish 

Turkey RBC preparation:

1. 4 ml of turkey blood is pipetted into a 15 ml conical and topped off with PBS.
2. Spin in a tabletop centrifuge at 800 rpm for 10 minutes.
3. Aspirate the supernatant without disturbing the blood cells.
4. Add 12 ml PBS and mix by inverting – do not vortex.
5. Spin at 800 rpm for 5 minutes and repeat wash two more times.
6. Aspirate supernatant after final wash and add enough PBS to make a 10% solution of red blood cells. This solution is useable for one week.
7. Make a final working solution of 0.5% RBCs in PBS. 

Viral Dilution and Assay:

1. A round-bottomed 96-well dish is preferred for this assay. Flat-bottomed plates will also work but need to be placed at an incline to develop.
2. To each well, add 50 μl PBS.
3. In the first column, add 50 µl of virus sample.
4. Mix each well and transfer 50 µl to the next well on its right. Repeat mixing and transferring 50µl down the length of the plate. Discard 50 µl from the last well into a bleach solution.
5. Add 50 µl of 0.5% red blood cell working solution to each well. Mix gently.
6. Leave at room temperature for 30-60 minutes to develop. Negative results will appear as dots in the center of round-bottomed plates. Positive results will form a uniformly reddish color across the well.
7. The virus’s HA titer is a simple number of the highest dilution factor that produced a positive reading.