A Night Out with Mycology – How Yeast and Fungi Add Culture to Our Lives

Cara N. Wilder, Ph.D.

It is a cool, crisp autumn evening. You are sitting in a downtown restaurant, gazing dreamily out the window, admiring the changing leaves and observing the occasional pedestrian hurrying by, bundled up from the chill. You begin to relax as you casually sip a glass of red wine, letting the smooth, rich liquid excite your taste buds and gradually warm your blood. In the background, a single violin begins to play a soft melody that sounds strangely familiar. As you ponder the origins of the musical piece, you are momentarily distracted by the waiter that has arrived at your table, presenting you with a warm Portobello sandwich. As you take your first bite, you savor every flavor; the subtle yeasty hue of the bun, the rich earthy tones of the Portobello mushroom, and the piquant bite of the bleu cheese dressing. You chuckle softly to yourself as you realize that your evening would not have been possible without Mycology.

Penicillium sp. Photo courtesy of the CDC

Yeast and fungi play a much larger role in our lives than commonly imagined by most individuals. For centuries, yeasts and fungi have been used in the generation of various food products and alcoholic beverages. In the preparation of the exquisite meal described above, yeast contributed to the production of the bread and wine, the Portobello mushroom is a fungus, and mold was used in the preparation of the bleu cheese dressing. Products such as bread, wine, and beer are commonly produced using various species of Saccharomyces, among other genera, which convert present fermentable sugars into ethanol and carbon dioxide. The flavor and consistency of these products are affected by the ingredients and the strain of yeast used. In contrast, bleu cheeses are commonly produced from cow’s milk, sheep’s milk, or goat’s milk cheeses that have cultures of the mold, Penicillium roqueforti or Penicillium glaucum. These molds give bleu cheeses their pungent flavor and characteristic blue tinge.

In addition to their role in fine dining, fungi have recently been implicated in shaping the musical world.  Fungal infections of trees caused by the species Physisporinus vitreus and Xylaria longipes, among others, are found to cause a decrease in wood density, ideal for creating a violin with superb acoustics.  Violins produced in this fashion, known colloquially as bioviolins, were found to be equivalent if not superior in sound quality as compared to Stradivarius violins. These latter violins are known to the musical world as the highest quality instruments made. In a recent sound quality experiment, over 90 individuals in a panel of 180 people ranked the bioviolin sound quality above that of the Stradivarius. However, only time will tell if this technique will continue to be used for the construction of violins or other wood-based instruments.

So, the next time you go out on the town for a nice meal or listen to music echoing wistfully from the body of a violin, think of Mycology. Remember, your microflora isn’t the only culture you have!

Tips for Aseptic Technique in the Laboratory – Biosafety Cabinets (Part 4 of 4)

Cara N. Wilder, Ph.D.

When working with hazardous or sterile materials, it is recommended that all associated procedures are performed in a biosafety cabinet. These apparatuses are enclosed, ventilated workspaces designed to protect laboratory personnel and materials from cross contamination during routine procedures. Generally, work spaces within the biosafety cabinet are protected through the use of a high-efficiency particulate air (HEPA) filtration method, which removes harmful microbes from the air.

There are three types of biological safety cabinets: Class I, Class II, and Class III.

Class I: These biological safety cabinets are open-front negative pressure systems with HEPA filtration systems. These biosafety cabinets provide protection for laboratory personnel and the environment, but will not provide product protection. These cabinets are often used to enclose specific equipment or procedures that may generate potentially hazardous aerosols.

Class II: These biological safety cabinets are open-front, ventilated, laminar-flow cabinets. These provide HEPA-filtered, recirculated airflow within the work space. Class II cabinets provide protection to laboratory personnel, the environment, and to products by drawing a curtain of sterile air over the products that are being handled. These cabinets are commonly used in microbiology laboratories working with non-infectious agents (Biosafety Level 1 or 2) as they protect the contained materials from extraneous airborne contaminants.

Class III: These biological safety cabinets are totally enclosed, ventilated systems with gas-tight construction. These cabinets are used through attached rubber gloves. Generally, the air supply is drawn into the cabinet through HEPA filters, and the exhaust air is filtered by two HEPA filters installed in a series. This biosafety cabinet system is commonly used with high-risk infectious agents (Biosafety Level 3, 4, or 5) to prevent the escape of aerosols.

Below, we describe some tips on how to properly use a Class I or Class II biosafety cabinet when working with non-infectious materials.

Keeping a Biosafety Cabinet Safe

·         Certify all biosafety cabinets upon installation

·         Routinely test the quality of airflow

·         Ensure the integrity of filters

·         Biosafety cabinets should be approved by the resident Biosafety officer

·         Ensure that all biosafety cabinets are routinely recertified

Biosafety Cabinet Sanitation

·         Clean work surfaces with 70% ethanol before and after use. When using 70% ethanol, ensure that there are no open flames nearby.

·         If the biosafety cabinet is equipped with germicidal UV lights, decontaminate work surfaces before and after use by turning on the UV light for at least 15 minutes. Never use the UV light while the biosafety cabinet is in use.

·         Routinely remove any biohazard waste from the biosafety cabinet.

·         Using an appropriate disinfectant, such as 70 % ethanol, wipe down the outer surface of all pipettes, pipette tip boxes, media, materials, etc. prior to placing them in the biosafety cabinet.

·         Always wear a clean lab coat and sterile gloves when working in a biosafety cabinet.

Proper use of a Biosafety Cabinet

·         Turn on the biosafety cabinet 15 minutes prior to use.

·         Only raise the biosafety cabinet sash to the recommended level, this will reduce disruption to the air flow as well as assist in the prevention of airborne contaminant entry.

·         When using a biosafety cabinet, limit the amount of movement in the cabinet and do not remove your arms. Additionally, limit the access to the area around the biosafety cabinet. This will reduce disruption to the airflow.

·         Do not use open flames within a biosafety cabinet. The resulting heat from the flame can disrupt the air flow provided by the biosafety cabinet, increasing the risk for contamination. Additionally, gas leaks associated with Bunsen burners or the use of an alcohol-based disinfectant near an open flame can result in fire or injury.

Stay clean, and happy culturing!

Using the pathogenic prowess of viruses to benefit the medical field

Cara N. Wilder, Ph.D.

For over a century, viruses have captivated scientific minds. These agents of destruction are often considered the border between biochemistry and biology, not truly alive in the classical sense nor are they unorganized amalgamations of protein and DNA. In actuality, viruses are metabolically inert, ultramicroscopic organisms with the inherent ability to cause disease by invading host cells and hijacking replicative machinery. It is this natural infection process that not only fascinates contemporary scientists but has allowed them to develop exciting viral-based technologies, including novel drug delivery methods and vectored vaccines, for the advancement of disease treatment and prevention.

When creating viral nanoparticles, the genomic material is
removed from the viral head and replaced with drugs.
Photo: CDC/ Doug Jordan

Viral nanoparticles are viruses whose genomic material has been removed and replaced with therapeutic drugs. Because viruses have naturally evolved the ability to cross the host membrane, they are an ideal candidate for drug packaging and the targeted delivery to cancer cells. To minimize toxic side effects, infection, and induced immune response, human viruses are not used in the production of viral nanoparticles. Rather, these drug delivery systems are engineered from plant, insect, and animal viruses. Plant viruses, such as the Cowpea mosaic virus, are ideal candidates for nanotechnology as they are easy to produce in large quantities, can self-assemble around a nanoparticle, and hold a substantial volume of drugs.

One of the major benefits to using viral nanoparticles for drug delivery is that targeting molecules can be easily attached to the viral surface, allowing for the directed binding to cancer cells. This specific binding prevents the harm or death of surrounding healthy cells, thus reducing possible side effects. Though viral nanoparticles have the potential to be a beneficial treatment, there are several complications associated with their use as a drug delivery system including immune rejection and potential toxicity. Regardless, viral nanoparticles have the capability to revolutionize the treatment of disease, creating a safe and specific form of drug delivery.

In addition to functioning as a drug delivery method, viruses can also be employed as vectors for vaccines. Generally, vaccines are the most effective and inexpensive prophylactic tool for the prevention of disease. Viral-based vaccines are traditionally employed as live attenuated viruses or as chemically inactivated viruses. Though these types of preparations have been shown to induce protective immunity in animal models, mutations in attenuated viruses or incomplete viral inactivation poses a serious risk to those who are vaccinated. Recombinant vectored vaccines, however, offer a live-vaccine approach that does not employ the complete pathogen. Instead, genetic material from the microbial target strain is inserted into the genome of harmless viral strains, allowing for the safe expression of microbial antigens.

Vectored vaccines can constructed to protect against a
wide variety of microbial diseases.
Photo: CDC/ Doug Jordan

Newcastle disease virus (NDV), a negative-sense avian virus, is a common template used for the development of recombinant vectored vaccines. Not only does NDV grow to high titers in many cell lines and eggs, it can elicit a strong immune response in vivo, is harmless to humans, and is commercially available. Additionally, NDV replicates in the cytoplasm of infected cells, thus eliminating the problem of genetic integration. To create a viral vaccine vector from NDV, genes encoding foreign target proteins are inserted into the NDV genome through recombination. Upon vaccination with the live virus, the foreign target protein is expressed within the host cell cytoplasm where it is then available for processing by the cellular antigen-processing machinery for immune presentation. As a result, cellular immunity is activated and neutralizing antibodies are generated.

Overall, the ability of viruses to cause devastating epidemics has left them greatly feared by the human population. However, with contemporary technologies such as viral-based drug delivery systems and vectored vaccines, viruses can now be regarded as tools in disease treatment and prevention. Perhaps with further advancement in viral-based technologies and applications, scientists will be able to harness the full benefit of these organisms, improving life where there was once death.

Tips for Aseptic Technique in the Laboratory -- Personal Protection and Cleanliness (Part 3 of 4)

Cara N. Wilder, Ph.D.

In a Microbiology laboratory, it is important to maintain the health and safety of all personnel. To avoid health risks, laboratory scientists should wear protective gear and be aware of any potential hazards nearby. Generally, protective wear serves two purposes; to protect the scientist from laboratory hazards and to shield the experiment from unintentional contamination. Below, we describe several mechanisms to maintain laboratory safety.


A scientist wearing a lab coat, disposable
gloves, and protective eye-wear

• Upon entering and exiting a lab, be sure to thoroughly wash your hands with anti-microbial soap.
• While in the lab, avoid touching your face or eyes. Further, do not handle make-up or contacts as they may become contaminated.
• Only enter the lab wearing closed-foot shoes. Open-toed shoes should never be worn in a laboratory as it leaves you susceptible to potential cuts or infection from broken glassware or sharp equipment.
• While performing an experiment, wear a clean, fitted laboratory coat. For additional protection, use a closed-front laboratory coat. Laboratory coats should never leave the lab, and should be cleaned frequently. Additionally, ensure that the sleeves of your coat are fitted so that they will not catch fire near an open flame, nor become entangled.
• When working with microorganisms, wear fitted, disposable gloves. Flakes of dry skin harbor bacteria, which may provide a source of contamination; wearing gloves will mitigate the risk. Additionally, disposable gloves decrease the risk of infecting any hand wounds.
• Pull back long hair or use a hair cover. Long hair is notorious for attracting dust, a potential source of contamination. Additionally, long hair could be a health hazard when working near an open flame. 
• Use protective eye-wear or a face shield when working with hazardous materials or cultures. If your eyes come in contact with microorganisms or harmful chemicals, wash your eyes for 15 minutes in an eye-wash and seek medical attention immediately.

Stay clean, and happy culturing!

Necrotizing fasciitis -- An indiscriminant killer

Cara N. Wilder, Ph.D.

Imagine one of the happiest moments of your life turning into a fierce battle against impending death. This is the harsh reality a South Carolina woman had to face in May as she went from becoming a new mother to one of the latest victims of necrotizing fasciitis. Only days after giving birth to twins, Lana Kuykendall was admitted to the hospital after her husband, a certified EMT, noticed a rapidly expanding bruise on her leg. After enduring almost 20 surgical procedures to remove necrotic flesh and reconstruct the surface of her leg via skin grafting, she is considered fortunate to be alive and in one piece¹.

Photomicrograph of Group A Streptococcus,
one of the leading causative agents of
necrotizing fasciitis. Photo provider: CDC

In the United States alone, there are approximately 250 cases of necrotizing fasciitis per year of which 20% are fatal. Recent news reports, such as that listed above, have highlighted the ever-growing concern associated with necrotizing fasciitis. This rare infection results from the subcutaneous invasion of one or more bacterial species that rapidly spread across the fascial plane, destroying surrounding muscles, nerves, fat, blood vessels, and skin. Thus far, many bacterial species, infamously dubbed as “flesh-eating bacteria” by the media, have been identified as causative agents of necrotizing fasciitis. These strains include group A Streptococcus, Klebsiella, Clostridium, E. coli, Staphylococcus aureus, and Aeromonas hydrophila². These aforementioned strains, however, do not truly consume flesh. Rather, many of these organisms rapidly destroy flesh via the release of toxins that inhibit the immune response, directly kill tissue, or indirectly cause tissue death via hypoxia³.

Unfortunately, early diagnosis of necrotizing fasciitis is often missed due to the lack of specific clinical features^{2, 3}. Generally, cases begin with an existing infection often found on the midsection or an extremity. This preliminary infection may stem from puncture wounds, surgical incisions, insect bites, or a minor break in the skin. Patients initially experience fever and chills accompanied by signs of inflammation, including swelling, erythema, and pain, at the site of infection. As the infection advances, the area of swelling spreads rapidly to the surrounding tissues resulting in ulceration, blisters, and excruciating pain. Without proper diagnosis and treatment at this later stage, patients often succumb to septic shock and death.

Presently, the first line of defense against this invasive infection is through intravenous administration of broad-spectrum antibiotics. Once the causative agent is identified, antibiotic susceptibility testing is performed to determine appropriate antibiotic coverage. However, because toxins produced by the invading bacterial horde can destroy soft tissue and reduce blood flow, antibiotic therapy may not be entirely effective. Therefore, amputation or the debridement of necrotic tissues is often required^{2, 3}. This latter treatment, though, is highly invasive and may lead to subsequent opportunistic infections as well as affect the quality of life.

Overall, necrotizing fasciitis is a destructive disease with very little associated information regarding predisposition, prompt diagnosis, or effective non-debilitating treatment. Currently, the best hope a patient has at survival and avoiding amputation is through early diagnosis. Without the timely identification of necrotizing fasciitis, Lana Kuykendall, as described above, may have lost her leg to the disease. Thus, only through public awareness and further characterization of this condition and its causative agents can we hope to advance medical practices, save lives, and protect the quality of life.

1.       CNN Report, “South Carolina mom with flesh-eating bacteria improves, faces rehabilitation”. http://articles.cnn.com/2012-06-21/us/us_south-carolina-flesh-eating-bacteria_1_necrotizing-fasciitis-flesh-eating-healthy-tissue?_s=PM:US; June 21, 2012.

2.       Centers for Disease Control, “Necrotizing Fasciitis: A Rare Disease, Especially for the Healthy”. http://www.cdc.gov/Features/NecrotizingFasciitis/ Page last updated June 18, 2012.

3.       MedicineNet.com, “Necrotizing Fasciitis (Flesh-Eating Disease)”. http://www.medicinenet.com/necrotizing_fasciitis/article.htm.

Tips for Aseptic Technique in the Laboratory -- Handling Microbial Cultures and Media (Part 2 of 4)

Cara N. Wilder, Ph.D.

When working with any microbial strain, propagation success depends heavily on the prevention of cross-contamination by other microorganisms. Sources of contamination can include non-sterile supplies, media, reagents, unclean work surfaces and incubators, airborne particles, and unclean gloves. Below, we describe some tips recommended by ATCC for handling microbial cultures and media.


Maintain a clean, uncluttered bench
Photo provider: ATCC

Maintain a Sterile Work Area

• Before and after use, disinfect all work surfaces with 70% ethanol. This is especially important after any spills.
• Maintain an uncluttered work space; all work surfaces should only contain equipment that is required for your experiment. 
• Ensure that you have all necessary supplies before beginning an experiment. Being prepared will reduce the likelihood of careless contamination.
• Work may be performed in a thoroughly sterilized biosafety cabinet. Biosafety cabinets can be sterilized via ultraviolet light in conjunction with 70% ethanol.



Use sterilized incubators and
equipment
Photo provider: ATCC

• Do not open windows or use fans that circulate outside air.  If possible, work in laboratory settings that have air vents covered with filters. This will prevent the contamination of cultures by airborne particles. 
• Frequently clean water baths used for thawing or warming media or solutions. 
• Routinely sterilize incubators used for microbial propagation. 

Handling Media

• Before and after use, sterilize the outside container of all media and reagents with 70% ethanol.  Also, do not leave containers of media open longer than necessary.
• Aliquot sterile solutions into smaller volumes whenever possible. If you are unsure of the sterility of your media, it is best to discard it immediately.
• Avoid pouring sterile liquids from one container to another, this increases the likelihood of aerosolization as well as media contamination. Rather, use filtered pipette tips for the aseptic transfer of media.  This will prevent contamination of the pipettor and any subsequent cross-contamination. 
• 

  

  Aseptically transfer media using a filtered pipette tip.
  This is best performed in a sterile environment such as a
  biosafety cabinet.
  Photo provider: ATCC

  Never mouth pipette. This poses a health risk for personnel as well as increases the risk of contamination.
• Always use sterile glass or disposable plastic pipettes to work with liquid media.  Use each pipette only once to avoid cross contamination. 
• Only unwrap sterile pipettes or open sterile media and petri dishes when you are ready to use them.
• Avoid sharing media and reagents with coworkers. 
• Always use separate bottles of media with each microbial strain, this will prevent cross contamination.

Handling Microbial Cultures

• Before working with media and microbial cultures, wipe your hands and work area with 70% ethanol.
• Ensure you are wearing appropriate protective clothing. This will protect you from the culture as well as reduce accidental culture contamination.
• Only use sterile glassware, equipment, media, and reagents. Check media for contamination by observing for turbidity.
• Handle only one microbial culture at a time. The risk of cross contamination or misidentification increases when more then one strain is handled at a time. 
• 

  

  If possible, handle microbial cultures within a biosafety cabinet
  Photo provider: ATCC

  When handling a microbial culture, work quickly and carefully in an environment that has minimal distractions.
• Avoid passaging and subculturing microbial strains too many times. Continually subculturing a strain increases both the risk of contamination as well as genetic drift.
• Inspect cultures daily for signs of contamination. 

Geomyces destructans -- An expose on North America's newest supervillain

Cara N. Wilder, Ph.D. 

B. Wayne, one of the many victims
of G. destructans
Photo Provider:  CDC/
Dr. Winkler and Dr. Sikes

Watch out Batman, there is a new supervillain in town!  This cold-loving fungus, who goes by alias Geomyces "the nose" destructans, is emerging in eastern North America as the top Chiroptera fungal pathogen.  Since its first documented appearance 6 years ago, this infectious supervillain has wreaked havoc across the eastern coast, indiscriminately killing millions of bats of at least six different species.

The nefarious activities of this cold-loving fungus were first documented in 2006 within a tourist cave near Albany, New York.  Dead and dying bats were found strewn on the cave floor as well as within four nearby caves, 30 km west of Albany.  As of 2010, G. destructans' malicious crime spree was found to have spread to an additional twelve states as well as across the Canadian border in Ontario and Quebec.  Recent scientific investigations have even suggested that G. destructans' criminal reach extends to Europe as part of an international pathogenic fungal syndicate with ties in Germany, Switzerland, and Hungary.     



Mug shot of Geomyces destructans
(Prisoner No. MYA-4855)
  Photo Authors: G. Wibbelt, A. Kurth, D. Hellmann,
M. Weishaar, A. Barlow, M. Veith, J. Pruger,
T. Gorfol, L. Grosche, F. Bontadina, U. Zophel,

H.P. Seidl, P.M. Cryan, and D.S. Blehert

G. destructans is a psychrophilic fungus that has known hide-outs in cool locations including caves, a popular bat hang-out.  It is in these dark lairs where this villainous fungus attacks its hibernating chiroptera victims!  G. destructans is known to attack and invade the facial skin of its sleeping victims, slowly causing ulcers and eventually death.  It can be identified by several distinguishing characteristics including asymmetrically curved conida and the inability to grow at temperatures above 24 degrees Celsius. 

In recent news, the G. destructans type strain was apprehended by the USGS National Wildlife Health Center and deposited into a secure ampule within ATCC's liquid nitrogen containment unit.  Scientists observing the captured fungus have already sequenced its genome (MYA-4855D), and are awaiting its formal annotation (http://www.atcc.org/CulturesandProducts/Microbiology/FungiandYeast/tabid/177/Default.aspx#Geo).   

We urge scientists to aid in the analysis of this fungal nemesis!  Start your research today, and end G. destructans reign of terror!  Any information provided could be the key to saving the lives of millions of bats!