SITE-DIRECTED MUTAGENESIS BY PCR AMPLIFICATION

 The first step in the mutagenesis of the GFP gene is to methylate the plasmid which carries the gene. This plasmid, pGLO, has been propagated in E. coli and then purified. We have done the methylation for you due to time constraints but the procedure is listed below. The methylase used transfers methyl groups from S-adenosylmethionine to cytosine residues occurring next to guanine. When DNA is methylated in this way, and then transformed into a wild-type strain of E. coli the DNA will be degraded. Thus after amplification of the plasmid with mutagenic primers, resulting in a mutagenized plasmid that is not methylated, only this new plasmid DNA will be replicated in the cell.

Methylation reaction

Mix together:

100 ng of the pGLO plasmid

1.6 μl of 10X methylation buffer

1.6 μl of 10X S-adenosylmethionine (SAM)

1 μl DNA methylase

water to 16 μl total volume

Incubate at 370C for 1 hour.

YOU WILL BEGIN AT THIS STEP

Mutagenesis reaction

Mix together:

4 μl methylated plasmid

1 μl of pGLO primer mix

45 μl PCR Supermix High Fidelity

Cycling parameters:

94 C for 2 min.

94 C for 30 sec.

55 C for 30 sec.

68 C for 6 min.

back to step 2 for 20 more cycles

68 C for 10 min.

After amplification is complete remove 17 μl of the PCR reaction to a clean microfuge tube, add 3 μl of tracking dye, and run on an agarose gel to verify amplification. Next, 4 μl of the reaction is transformed into E.coli DH5 and plated on a rich media containing ampicillin and arabinose. The ampicillin selects for the plasmid and the arabinose induces the transcription of the GFP gene. It is important to keep the cells on ice and chilled until the heat shock in step 4. Never vortex competent cells.

1. Put 4 μl of each ligation reaction in a sterile 1.5 ml tube and place it on ice. Thaw competent cells on ice.

2. Transfer 50 μl of competent DH5α into each tube and gently mix by tapping the tube.

3. Incubate tubes on ice for 15 min.

4. Heat-shock the cells for 3 min. at 42°C.

5. Return tubes to ice for 2 min.

6. Add 900 ml LB broth and incubate at 37°C for 1 hour.

7. Spin culture, remove most of the media, resuspend pellet in remaining media and plate on LB + 100 μg/ml Ampicillin + 0.6% Arabinose

ISOLATION OF GENOMIC DNA FROM GRAM NEGATIVE BACTERIA

You will be given a 1.5 ml microcentrifuge tube containing a pellet of Escherichia coli, strain BW30270. This strain is a K-12 or laboratory strain of E.coli. The pellets are made up of cells from 3 ml of overnight growth in a rich medium.

1. Add 600 μl of Nuclei Lysis Solution. Gently pipet up and down until the cells are resuspended.

2. Incubate at 80°C for 5 minutes to lyse the cells; then cool to room temperature.

3. Add 3μl of RNase Solution to the cell lysate. Invert the tube 2–5 times to mix.

4. Incubate at 37°C for 30 minutes. Cool the sample to room temperature.

5. Add 200 μl of Protein Precipitation Solution to the RNase-treated cell lysate.

6. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution with the cell lysate, be sure that the two solutions have completely mixed. Do not over-vortex or you risk shearing the chromosome.

7. Incubate the sample on ice for 5 minutes. Centrifuge at 13,000 rpm for 10 minutes.

8. Transfer the supernatant containing the DNA to a clean 1.5ml micro-centrifuge tube containing 600μl of room temperature isopropanol. Do not carry over any flecks of the precipitate.

9. Gently mix by inversion until the thread-like strands of DNA form a visible mass. If you do not see threads but a general whitish appears mix well and continue.

10. Centrifuge at 13,000 rpm for 5 minutes.

11. Carefully pour off the supernatant and drain the tube on clean absorbent paper. Add 600 μl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.

12. Centrifuge at 13,000 rpm for 2 minutes. Carefully aspirate the ethanol from the tube with a pipet. Be careful not to suck up your pellet.

13. Drain the tube on kimwipes and allow the pellet to air-dry for 10–15 minutes.

14. Add 100μl of sterile water to the tube and rehydrate the DNA.

Introduction to Virology

A virus is an obligate intracellular parasite containing genetic material surrounded by a protein Virus particles can only be observed by an electron microscope

Viral Properties

1. Inert  (nucleoprotein ) filterable Agents
2. Obligate intracellular parasites
3. Cannot make energy or proteins independent of a host cell
4. Genome is RNA or DNA but not both.
5. Have a naked capsid or envelope with attached proteins
6. Multiply by a complex process, not by binary fission. 
7. Non-living entities?? 

Terms & Definitions in Virology

• Capsid: The protein shell, or coat, that encloses the NA genome.
• Capsomeres: Morphologic units of Capsid. Capsomeres represent clusters of polypeptides
• Defective virus: A virus particle that is functionally deficient in some aspect of replication.
• Envelope: A lipid-containing membrane that surrounds some virus particles. It is acquired during viral maturation by a budding process through a cellular membrane.
• Peplomers: virus-encoded glycoproteins are exposed on the surface of the envelope.
• Nucleocapsid: The protein-nucleic acid complex representing the packaged form of the viral genome.
• Virion: The complete virus particle. In some instances (eg, papillomaviruses, picornaviruses), the virion is identical with the nucleocapsid. In more complex virions (herpesviruses, orthomyxoviruses), this includes the nucleocapsid plus a surrounding envelope. This structure, the virion, serves to transfer the viral nucleic acid from one cell to another.   

The size of viruses

How stratospheric life is teaching us about the possibility of extreme life on other worlds

The presence of microbial life in Earth’s stratosphere is not only opening up a new arena in which to study extremophiles but is increasing the range of possible environments in which we may find life on other planets. This is the conclusion of a new study that summarizes what we know about stratospheric life so far.

The stratosphere is the atmospheric zone that lies directly above the dynamic troposphere where we live, but it is mostly a mystery when it comes to the life that exists there.

You might not realize it when you’re starting out a plane window (we fly through the lowest levels of the stratosphere when we’re cruising over 35,000 feet), but there are all kinds of microorganisms out there, according to Professor Shiladitya DasSarma, who is a microbiologist at the University of Maryland School of Medicine, the USA, and a co-author on the new study, which is published in the journal Current Opinion in Microbiology.

“Generally, people don’t think of microbes being airborne,” he tells Astrobiology Magazine. “But there’s a saying in microbiology: Everything is everywhere.”

However, there are “very few studies at the present time” that look at the atmospheric biome. Part of the issue is that there is a low density of cells in a large volume of air. But when you look at it globally, the numbers are significant: 1021 is the current estimate for the number of cells lifted annually into the atmosphere.

Still, the space involved is vast: “When you’re talking about the entire atmosphere of a planet, how do you do a survey of that?” asks Priya DasSarma, a research scientist also from the University of Maryland and the study’s lead author. She suggests it would have to be a community exercise with a long timeline, which would eventually result in what she calls an ‘Atlas of Stratospheric Microbes’.

“A program like that would be incredibly productive and interesting and worthwhile,” she says, not only for what it could tell us about life on Earth but also how cells could survive and even adapt to life on other planets. That has implications when it comes to planetary protection (not exposing other planets to terrestrial germs), and to astrobiology more generally.

“When we measure the response of terrestrial life in extreme environments on Earth, we can learn more about habitability across the Solar System and where to refine the search for life elsewhere,” says Dr. David J. Smith, a senior microbiologist in the Space Biosciences Division at NASA’s Ames Research Center.

Extreme environment 

Conditions in the stratosphere are brutal – it’s a dry, cold, hypobaric (i.e. low pressure), ultraviolet-drenched environment, which is why it serves as an apt analog to life on other worlds with similar conditions.

“The temperatures, UV, and dryness are similar to Mars, so it’s a great proxy,” says Shiladitya DasSarma. Nevertheless, life persists. Bacteria and fungi usually perish in this kind of environment, but those that survive do so via a few strategies. For instance, forming spores is a tried-and-true way to protect genetic material.

Yet even non-spore-forming extremophiles have mechanisms to protect themselves. “There’s a wide variety of stress-survival mechanisms,” says Shiladitya DasSarma. “For UV, a number of [extremophiles] have DNA damage repair mechanisms. Others have additional, more quiescent methods, like extreme halophiles that can survive very low-water situations because their proteins are designed to hold onto whatever small amount of water is present.”

Contaminating other worlds 

If life can survive the conditions in the stratosphere, perhaps life can also survive in space. When it comes to microbes hitchhiking on interplanetary spacecraft, it’s going to be increasingly important that we know which of these bacteria, archaea, or fungi can survive, since we know from the stratosphere studies that cold temperatures, UV radiation, and other factors won’t kill every last cell.

Currently, space agencies including NASA have a mandate not to expose other planets to Earth’s microfauna, so precautions are taken before launching landers. In most cases, there’s not likely to be much that will remain alive after a spacecraft has been doused in cosmic rays. However, we know from experience how hardy invasive species on Earth can be – there’s a reason life is “everywhere” on Earth.

“We know Mars is a dusty planet and spacecraft coated in dust might shade some microbial hitchhikers,” says Smith, who published a paper in 2017 examining this idea. “Also, a portion of bioburden [the number of microbes surviving on spacecraft] are embedded deep inside the spacecraft’s hardware where they are protected from radiation, substantially reducing or completely eliminating the effects of UV.” With just minimal protection, microbes can use the same strategies that allow them to survive in the stratosphere – like DNA repair of UV damage, or water storage – to stay alive far from Earth.

It’s important to keep in mind that surviving does not necessarily mean thriving. Just because an organism makes it to, say, Mars, doesn’t mean it will be viable and reproduce. That’s why knowing more about extremophiles, particularly those in Earth’s stratosphere, is key.

Conversely, at some point, we may actually want some of these microorganisms to thrive because good bacteria are going to be important partners for us when we set up human colonies. “If we want to go to Mars and inhabit it, we are going to want to bring whatever microbes and microbes [i.e. larger lifeforms] with us that we need to survive there,” says Priya DasSarma. “But we don’t want to bring anything that contaminates or destroys the environment that we’re going to.”

Knowing how and why tough organisms persist in the stratosphere above our heads will be important when it comes to protecting the planets that we explore in the short term. Meanwhile, looking farther into the future, those same extreme lifeforms could eventually help us to survive on other worlds as we expand out into the Galaxy.

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. 

Bacteria Of Medical Importance

Bacteria of Medical Importance

Historically, bacteria have been the cause of some of the most deadly diseases and widespread epidemics of human civilization. Although smallpox and malaria, diseases caused by other microbes, may have killed more humans than bacterial diseases, bacterial diseases such as tuberculosis, typhus, plague, diphtheria, typhoid fever, cholera, dysentery, and pneumonia have taken a mighty toll on humanity. Water purification, immunization (vaccination) and modern antibiotic treatment continue to reduce the morbidity and the mortality of bacterial disease in the Twenty-first Century, at least in the developed world where these are acceptable cultural practices. However, many new bacterial pathogens have been recognized in the past 25 years and many "old" bacterial pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis, have emerged with new forms of virulence and new patterns of resistance to antimicrobial agents.

Most of the bacterial pathogens of humans are classified as Gram-positive or Gram-negative, some notable exceptions being the mycoplasmas, chlamydiae, spirochetes and the mycobacteria. In this article, the major pathogens of humans are organized into natural groups based on bacteriological criteria, rather than on the basis of the affected organ, mode of transmission, or type of disease. This goes with being written by a bacteriologist.

Spirochetes

The spirochetes are a phylogenetically distinct group of bacteria which have a unique cell morphology and mode of motility. Spirochetes are very thin, flexible, spiral-shaped procaryotes that move by means of structures called axial filaments or endo-flagella. The flagellar filaments are contained within a sheath between the cell wall peptidoglycan and an outer membrane. The filaments flex or rotate within their sheath which causes the cells to bend, flex and rotate during movement. Most spirochetes are free-living (in muds and sediments), or live in associations with animals (e.g. in the oral cavity or GI tract). A few are pathogens of animals Treponema pallidum is the agent of syphilis, a sexually transmitted disease, and Borrelia burgdorferi causes Lyme Disease. which is transmitted by the bite of the deer tick.

Spirilla and other curved bacteria

Spirilla are Gram-negative bacteria with a helical or spiral shape. Their metabolism is respiratory and never fermentative. Unlike spirochetes, they have a rigid cell wall and are motile by means of ordinary polar flagella. Two important pathogens of humans are found among the spiral forms. Campylobacter jejuni is the cause of bacterial diarrhea, especially in children. The bacterium is transmitted via contaminated food, usually undercooked poultry or shellfish, or untreated drinking water. Helicobacter pylori are able to colonize the gastric mucosal cells of humans, i.e., the lining of the stomach, and it has been well established as the cause of peptic ulcers and there is strong evidence for its involvement in adenocarcinoma.

Vibrios

The term vibrio refers to a Gram-negative bacterium which has the cell shape of a curved rod or a comma. Members of the genus Vibrio are common bacteria in aquatic environments, especially marine environments. They have structural and metabolic properties that overlap with both the enterics and the pseudomonads. Vibrios are facultative (grow in the presence or absence of O2), like enterics, but they have polar flagella, are oxidase-positive, and degrade sugars in the same manner as the pseudomonads. In aquatic habitats, they overlap with the pseudomonads in their ecology, although pseudomonads favor freshwater and vibrios prefer salt water. Some marine vibrios are bioluminescent (they emit light) and some are symbionts of fish, squid and other marine life. Vibrio cholera causes an epidemic or Asiatic cholera which, untreated, is one of the most rapidly fatal infectious diseases known. The pathology is related to diarrheal diseases caused by the enteric bacteria, except it is relentless, and a patient can die rapidly from dehydration. The cholera toxin, which is the classic model of a bacterial enterotoxin, is also produced by some strains of E. coli.

The Gram-negative aerobic rods and cocci

The name refers to Gram-negative bacteria phenotypically related to members of the genus Pseudomonas. Their metabolism is respiratory and never fermentative. Important human pathogens include Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, Bordetella pertussis, Haemophilus influenzae, Legionella, Brucella and Francisella, and a few others. Many bacteria in this physiological group are free-living in soil and water, and they play an important role in decomposition, biodegradation, and the C and N cycles. Also, many bacteria which are pathogens of plants are found in this group, including Pseudomonas, Xanthomonas, and Agrobacterium.

Pseudomonas aeruginosa is the quintessential opportunistic pathogen of humans. It is a leading cause of hospital-acquired infections (nosocomial infections), and it is difficult to erradicate due to its resistance to most antimicrobial agents. There is probably no tissue that cannot become infected by Pseudomonas if the host defenses are weakened, and it is difficult to erradicate due to it's resistance to antimicrobial agents. It is usually involved in soft tissue infections, urinary tract infections and pneumonia.

Whooping cough (or pertussis) is caused by Bordetella pertussis. The disease is particularly serious in infants and young children and has a high mortality rate. Whooping cough is controlled by vaccination with the acellular pertussis vaccine, which is usually given in association with diphtheria, tetanus and sometimes H. influenzae type b (Hib), as part of the childhood immunization program in the U.S.

Legionaires' pneumonia is caused by Legionella pneumophila. This pneumonia, and the bacterium, were not discovered until 1976, when there was an outbreak of disease at a Legionaire's meeting in Philadelphia. It took several months to find, culture and grow the

bacterium. The incident was a wake-up call to public health officials that there were probably a lot of disease-producing bacteria outthere that they know nothing about.

Neisseria gonorrhoeae causes the sexually-transmitted disease gonorrhea, and Neisseria meningitidisis the agent of meningicoccal meningitis. The Neisseriae are discussed below with the Pyogenic Cocci.

Haemophilus influenzae is also a cause of meningitis, but the icidence of the disease has declined rapidly with the use of the Hib vaccine which began in 1994. Haemophilus is sometimes involved in infections of the upper respiratory tract, particularly the sinuses.

Brucellosis is a chronic debilitating infection in humans associated with reproductive failure in domestic animals. Person-to-person transmission of brucellae is extremely rare. Brucella abortus is the species usually involved in human disease. The primary reservoir of the organism is in cattle, although bison are sometimes wrongfully accused.

Enterics

Enteric bacteria are Gram-negative rods with facultative anaerobic metabolism that live in the intestinal tracts of animals in health and disease. This group consists of Escherichia coli and its relatives, the members of the family Enterobacteriaceae. Enteric bacteria are related phenotypically to several other genera of bacteria such as Pseudomonas and Vibrios. Generally, a distinction can be made on the ability to ferment glucose; enteric bacteria all ferment glucose to acid end products while similar Gram-negative bacteria (e.g. pseudomonads) cannot ferment glucose. Because they are consistent members of the normal flora humans, and because of their medical importance, an extremely large number of enteric bacteria have been isolated and characterized.

Escherichia coli is, of course, the type species of the enterics. E. coli is such a regular inhabitant of the intestine of humans that it is used by public health authorities as an indicator of fecal pollution of drinking water supplies, swimming beaches, foods, etc. E. coli is the most studied of all organisms in biology because of its occurrence, and the ease and speed of growing the bacteriium in the laboratory. It has been used in hundreds of thousands of experiments in cell biology, physiology, and genetics, and was among the first cells for which the entire chromosomal DNA base sequence (genome) was determined. In spite of the knowledge gained about the molecular biology and physiology of E. coli, surprisingly little is known about its ecology, for example, why it consistently associates with humans, how it helps its host, how it harms its host, etc. A few strains of E. coli are pathogenic (one is now notorious, strain 0157:H7, that keeps turning up in raw hamburger headed for a fast-food restaurants). Escherichia coli causes intestinal tract infections (usually acute and uncomplicated, except in the very young ) or uncomplicated urinary tract infections and neonatal meningitis.

The enteric group also includes some other intestinal pathogens of humans such as Shigella dysenteriae, cause of bacillary dysentery, and Salmonella enteritidis, cause of food poisoning and gastroenteritis. Salmonella typhi, which infects via the intestinal route, causes typhoid fever. Some bacteria that don't have an intestinal habitat resemble E. coli in enough ways to warrant inclusion in the enteric group. This includes Proteus, a common saprophyte of decaying organic matter and Yersinia pestis, which causes bubonic plague. Also classified as an enteric is Erwinia, a pathogen of plants that causes fireblight in pear and apple trees and soft rot of carrots and potatoes.

Pyogenic Cocci

The pyogenic cocci are spherical bacteria that cause various suppurative (pus-producing) infections in animals. Included are the Gram-positive cocci Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae,and the Gram-negative cocci, Neisseria gonorrhoeae and N. meningitidis. In terms of their phylogeny, physiology and genetics, these genera of bacteria are unrelated to one another. They share a common ecology, however, as parasites of humans.

The Gram-positive cocci are the leading pathogens of humans. It is estimated that they produce at least a third of all the bacterial infections of humans, including strep throat, pneumonia, food poisoning, various skin diseases and severe types of septic shock. The Gram-negative cocci, notably the neisseriae, cause gonorrhea and meningicoccal meningitis.

Two species of Staphylococcus live in association with humans: Staphylococcus epidermidis which lives normally on the skin and mucous membranes, and Staphylococcus aureus which may occur normally at various locales, but in particular on the nasal membranes (nares). S. epidermidis is rarely a pathogen and probably benefits its host by producing acids on the skin that retard the growth of dermatophytic fungi.
S. aureus always has the potential to cause disease and so is considered a pathogen. Different strains of S. aureus differ in the range of diseases they can cause, including boils and pimples, wound infections, pneumonia, osteomyelitis, septicemia, food intoxication, and toxic shock syndrome. S. aureus is the leading cause of nosocomial (hospital-acquired) infections by Gram-positive bacteria. Also, it is notoriously resistant to penicillin and many other antibiotics. Recently, a strain of S. aureus has been reported that is resistant to all known antibiotics in clinical usage, which is a grim reminder that the clock is ticking on the lifetime of the usefulness of current antibiotics in treatment of infectious disease.

Staphylococcus aureus is a successful bacterial pathogen because it has a very wide range of virulence determinants (structural, biochemical or genetic features that allow the bacterium to cause disease), and it occurs as normal flora of humans (on skin, nasal membranes and the GI tract), which ensures that it is readily transmitted from one individual to another.

Streptococcus pyogenes, more specifically the beta-hemolytic group A streptococci, like S. aureus, causes an array of suppurative diseases and toxinoses (diseases due to the production of a bacterial toxin), in addition to some autoimmune or allergic diseases. S. pyogenes is occasionally found as normal flora in the upper respiratory tract(<15% of individuals), but it is the main streptococcal pathogen for man, most often causing tonsillitis or strep throat. Streptococci also invade the skin to cause localized infections and lesions, and produce toxins that cause scarlet fever and toxic shock. Sometimes, as a result of an acute streptococcal infection, anomalous immune responses are started that lead to diseases like rheumatic fever and glomerulonephritis, which are called poststreptococcal
sequelae. Unlike the staphylococci, the streptococci have not developed widespread resistance to penicillin and the other beta lactam antibiotics, so that the beta lactams remain drugs of choice for the treatment of acute streptococcal infections.

Streptococcus pneumoniae is the most frequent cause of bacterial pneumonia in humans. It is also a frequent cause of otitis media (infection of the middle ear) and meningitis. The bacterium colonizes the nasopharynx and from there gains access to the lung or to the eustachian tube. If the bacteria descend into the lung they can impede engulfment by alveolar macrophages if they possess a capsule which somehow prevents the engulfment process. Thus, encapsulated strains are able to invade the lung and are virulent (cause disease) and noncapsulated strains, which are readily removed by phagocytes, are nonvirulent.
The Neisseriae cause gonorrhea and meningitis. Neisseriaceae is a family of Gramnegative bacteria with characteristics of enterics and pseudomonads. The neisseriae are small, Gram-negative cocci usually seen in pairs with flattened adjacent sides. Most neisseriae are normal flora or harmless commensals of mammals living on mucous membranes. In humans they are common residents of the throat and upper respiratory tract. Two species are primary pathogens of man, Neisseria gonorrhoeaeand Neisseria meningitidis.

Neisseria gonorrhoeae is the second leading cause of sexually-transmitted disease in the U.S., causing over 300,000 cases of gonorrhea annually. Sometimes, in females, the disease may be unrecognized or asymptomatic such that an infected mother can give birth and unknowingly transmit the bacterium to the infant during its passage through the birth canal. The bacterium is able to colonize and infect the newborn eye resulting neonatal ophthalmia, which may produce blindness. For this reason (as well as to control Chlamydia which may also be present), an antimicrobial agent is usually added to the newborn eye at the time of birth.

Neisseria meningitidis is an important cause of bacterial meningitis, an inflammation of the meninges of the brain and spinal cord. Other bacteria that cause meningitis include Haemophilus influenzae,Staphylococcus aureus and Escherichia coli. Meningococcal meningitis differs from other causes in that it is often responsible for epidemics of meningitis. It occurs most often in children aged 6 to 11 months, but it also occurs in older children and in adults. Meningococcal meningitis can be a rapidly fatal disease, and untreated meningitis has a mortality rate near 50 percent. However, early intervention with antibiotics is highly effective, and with treatment most individuals recover without
permanent damage to the nervous system.

Endospore-forming bacteria

Endospore-forming bacteria produce a unique resting cell called an endospore. They are Gram-positive and usually rod-shaped, but there are exceptions. The two medically important genera are Bacillus, the members of which are aerobic spore formers in the soils, and Clostridium, whose species are anaerobic spore formers of soils, sediments and the intestinal tracts of animals. Some sporeformers are pathogens of animals, usually due to the production of powerful toxins. Bacillus anthracis causes anthrax, a disease of domestic animals (cattle, sheep, etc.), which may be transmitted to humans. Bacillus cereus causes food poisoning. Clostridium botulimum causes botulism, a form of food poisoning, and Clostridium tetani is the agent of tetanus. Clostridium perfringens causes food poisoning, anaerobic wound infections and gas gangrene, and Clostridium difficile causes asevere form of colitis called pseudomembranous colitis. Whenever the spore-formers act as pathogens, it is not uncommon or surprising that their spores are somehow involved in transmission or survival of the organism between hosts.

Listeria monocytogenes is a Gram-positive rod-shaped bacterium related to bacillus and Clostridium but it does not form endospores. Listeria monocytogenes is the agent of listeriosis, a serious infection caused by eating food contaminated with the bacteria.
Listeriosis has recently been recognized as an important public health problem in the United States. The disease affects primarily pregnant women, newborns, and adults with weakened immune systems

Actinomycetes and related bacteria

The actinomycetes are not thought of as pathogenic bacteria, but two of their relatives are among the most important pathogens of humans, these being the agents of tuberculosis and diphtheria. Actinomycetes are a large group of Gram-positive bacteria that usually grow by filament formation, or at least show a tendency towards branching and filament formation. Many of the organisms can form resting structures called spores, but they are not the same as endospores. Branched forms superficially resemble molds and are a striking example of convergent evolution of a procaryote and a eukaryote together in the soil habitat. Actinomycetes such as Streptomyces have a world-wide distribution in soils.
They are important in aerobic decomposition of organic compounds and have an important role in biodegradation and the carbon cycle. Actinomycetes are the main producers of antibiotics in industrial settings, being the source of most tetracyclines, macrolides (e.g. erythromycin), and aminoglycosides (e.g. streptomycin, gentamicin, etc.).
Two genera of bacteria that are related to the actinomycetes, Corynebacterium and Mycobacterium, contain portant pathogens of humans: Otherwise, many nonpathogenic mycobacteria and corynebacteria live in normal associations with animals.

Mycobacterium tuberculosis is the etiologic agent of tuberculosis (TB) in humans. Tuberculosis is the leading cause of death in the world from a single infectious disease. Mycobacterium tuberculosis infects 1.7 billion people/year which is equal to 33% of the entire world population. The bacterium is responsible for over 3 million deaths/year.
After a century of decline in the United States, tuberculosis is increasing, and multiple drug-resistant strains have emerged. This increase in cases is attributable to changes in the social structure in cities, the HIV epidemic, and patient failure to comply with treatment programs. A related organism, Mycobacterium leprae, causes leprosy.

The genus Corynebacterium consists of a diverse group of bacteria including animal and plant pathogens, as well as saprophytes. Some corynebacteria are part of the normal flora of humans, finding a suitable niche in virtually every anatomic site. The best known and most widely studied species is Corynbacterium diphtheriae, the causal agent of diphtheria. The study of Corynebacterium diphtheriae traces closely the development of medical microbiology, immunology and molecular biology. Many contributions to these fields, as well as to our understanding of host-bacterial interactions, have been made studying diphtheria and the diphtheria toxin.

Rickettsias and chlamydiae are two unrelated groups of bacteria that are obligate intracellular parasites of eukaryotic cells. Rickettsias cannot grow outside of a host cell because they have leaky membranes and are unable to obtain nutrients in an extracellular habitat. Chlamydiae are unable to produce ATP in amounts required to sustain metabolism outside of a host cell and are, in a sense, energy-parasites.

Rickettsias occur in nature in the gut lining of arthropods (ticks, fleas, lice, etc.). They are transmitted to vertebrates by an arthropod bite and produce diseases such as typhus fever, Rocky Mountain Spotted Fever, Q fever and ehrlichiosis.

Chlamydiae are tiny bacteria that infect birds and mammals. They may colonize and infect tissues of the eye and urogenital tract in humans. Chlamydia trachomatis causes several important diseases in humans: chlamydia, the most prevalent sexually transmitted disease in the U.S., trachoma, a leading cause of blindness worldwide, and lymphogranuloma venereum. Chlamydia pneumoniae is a cause of pneumonia and has been recently linked to atherosclerosis.

Mycoplasmas are a group of bacteria that lack a cell wall. The cells are bounded by a single triple-layered membrane. They may be free-living in soil and sewage, parasitic inhabitants of the mouth and urinary tract of humans, or pathogens in animals and plants. In humans, Mycoplasma pneumoniae causes primary atypical pneumonia, also called walking pneumonia.

Identification of Bacteria by Different Methods

Bacterial Identification techniques