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Posted by Karen on August 14th, 2018  ⟩  0 comments

Making sure your experiment goes right is a top priority because it saves time, money and prevents the overall frustration of the job. In many DNA extraction protocols, the use of proteinase K is an important step because of its ability to digest harmful nucleases, but how much to use, when to use it and for how long can sometimes be a mystery. In this article, we untangle 5 common proteinase K questions that relate closely to extraction methods. While we hope that this article serves as a helpful guide in your work, it is critical to do additional research to make sure your methods are perfectly matched to the type of work you’re doing.

To view a printable proteinase K digestion table, click here or scroll down for the PDF.

Proteinase K Protocol and Digestion Guide



When is proteinase K used?

Proteinase K is used mostly in DNA and RNA extraction protocols. You’ll often find the proteinase K step within the lysis section of the protocol. For example, in the nucleic acid extraction protocol, proteinase K is added to cell lysate and then an incubation period follows to ensure a complete digestion.

To prevent potential digestion of your samples, proteinase K is inactivated after incubation. The common temperature for inactivation is 95°C.

Even in the typical mouse-tail protocol, proteinase K is regularly used to inhibit harmful nucleases. And the addition of proteinase K occurs during the digestion step. The use of EDTA is also suggested to help the inactivation of nucleases by inhibiting Mg2+ dependent nucleases.



How do I know if digestion has happened?

Usually, the biggest tell that complete digestion has occurred is that you should see a clear lysed cell solution. If you are not seeing a clear solution after the initial digestion period, extend your incubation time.

Be very careful with this. If you are using a faster method for isolation, especially involving higher volumes of proteinase K, you’ll need to pay close attention during proteinase K digestion. A longer digestion may cause degradation of your DNA.



How long of an incubation time should I use?

The incubation period with proteinase K is going to depend primarily on the type of sample you’re working with. After doing quite a bit of research, here is the range of times we found for different cell and tissue samples. Please keep in mind that your experiment may have different requirements or variables that could greatly influence digestion times, and therefore we strongly encourage you to do your own research before carrying out your work.

Formalin-Fixed Paraffin-Embedded Tissues Digest for several hours to overnight.

*Note: Formalin-Fixed Paraffin-Embedded Tissues commonly appears in the abbreviated form, FFPE. This is a method for tissue preservation (another long-term tissue preservation method is with frozen tissue).

  • Bacteria – Digest with proteinase K between 1-3 hours. Digestion temperature may also influence how long your digestion should take.
  • Mammalian cells – There are several papers out there with a wide range of stated digestion times – as little as 1 hour and as long as twelve hours. This is partly due to experimental objectives and the type of cells used. Digestion temperature and proteinase K volumes also have some influence.



What temperature should I use for proteinase K digestion?

Digestion temperatures also vary with the type of sample you’re working with. Once again, we offer a guide on this, but strongly encourage you to do more research to optimize all conditions before proceeding with your experiment.

  • FFPE Tissue – Digestion temperatures 55-56°C. Most articles are fairly consistent with that temperature range.
  • Bacteria – Digestions are often carried out at 55°C. Some articles, however, did state a 37°C digestion temperature. Keep in mind the requirements of the type of sample you’re working with and other factors of your experiment.
  • Mammalian – Articles greatly varied in digestion temperatures. Shorter digestion periods usually correlated with higher temperatures (optimal proteinase K digestion temperatures for mammalian cells range between 50-65°C). Articles with digestions taking place for several hours to overnight usually suggested 37°C. Several other factors can impact the digestion temperature such as cell type (blood, buccal, etc.) and molecular weight.



How much proteinase K do I use?

The amount of proteinase K you need for successful digestion is going to depend on many factors: the protocol you’re using, the type of sample you’re working with, the conditions of your experiment, etc. Typically, 10-20 µl of proteinase K are used in experiments, with stock proteinase k stock concentrations usually around 20 mg/ml.

Something else to keep in mind is that some methods require a second digestion step (usually those involving tissue samples). Weaker, second digestions usually call for a lower volume and a different digestion period.

For more proteinase K tips, visit the product page for a list of related literature, or check out our articles on common questions about proteinase K and proteinase K activity.



References


Bielawski, K., Zaczek, A., Lisowska, U., Dybikowska, A., Kowalska, A., & Falkiewicz, B. (2001). The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis. International Journal of Molecular Medicine.doi:10.3892/ijmm.8.5.573Biase, F. H., Franco, M. M., Goulart, L. R., & Antunes, R. C. (2002). Protocol for extraction of genomic DNA from swine solid tissues. Genetics and Molecular Biology,25(3), 313-315. doi:10.1590/s1415-47572002000300011

Chen, Z. (n.d.). Zhibin’s Protocol of Tissue Processing for PCR genotyping. Zhejiang University. Retrieved July 17, 2018, from http://person.zju.edu.cn/attachments/2012-05/07-13...

Derua, Y. A., Alifrangis, M., Hosea, K. M., Meyrowitsch, D. W., Magesa, S. M., Pedersen, E. M., & Simonsen, P. E. (2012). Change in composition of the Anopheles gambiae complex and its possible implications for the transmission of malaria and lymphatic filariasis in north-eastern Tanzania. Malaria Journal,11(1), 188. doi:10.1186/1475-2875-11-188

DNA EXRACTING FROM TOE PADS OR FEATHERS USING GENECLEAN II. (2005). Lougheed Genetics Laboratory Manual, Queens University. Retrieved July 17, 2018, from http://www.ispybio.com/search/protocols/dna_extractios_etc.pdf

DNA Isolation From Blood or Tissue Using Phenol/Chloroform. (2005). Lougheed Genetics Laboratory Manual, Queens University. Retrieved July 17, 2018, from http://www.ispybio.com/search/protocols/dna_extrac...

Fan, H., & Gulley, M. L. (2001). DNA Extraction from Fresh or Frozen Tissues. Molecular Pathology Protocols,5-10. doi:10.1385/1-59259-081-0:5

Feil, W., Feil, H., & Copeland, A. (2012, November 12). Bacterial genomic DNA isolation using CTAB [Web log post]. Retrieved July 16, 2018, from https://www.researchgate.net/profile/Ali_Mahmoudpo...

Goldenberger, D., Perschil, I., Ritzler, M., & Altwegg, M. (1995). A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. Genome Research,4(6), 368-370. doi:10.1101/gr.4.6.368

Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H., & Lange, T. D. (1999). Mammalian Telomeres End in a Large Duplex Loop. Cell,97(4), 503-514. doi:10.1016/s0092-8674(00)80760-6

Gross-Bellard, M., Oudet, P., & Chambon, P. (1973). Isolation of High-Molecular-Weight DNA from Mammalian Cells. European Journal of Biochemistry,36(1), 32-38. doi:10.1111/j.1432-1033.1973.tb02881.x

Hrncirova, K., Lengerova, M., Kocmanova, I., Racil, Z., Volfova, P., Palousova, D., . . . Mayer, J. (2010). Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis. Journal of Clinical Microbiology,48(9), 3392-3394. doi:10.1128/jcm.01109-10

Lum, A., & Marchand, L. (1998). A Simple Mouthwash Method for Obtaining Genomic DNA in Molecular Epidemiological Studies. Cancer Epidemiology, Biomarkers & Prevention,7, 719-724. Retrieved July 17, 2018, from http://cebp.aacrjournals.org/content/cebp/7/8/719....

Meulenbelt, I., Droog, S., Trommelen, G., Boomsma, D., & Slagboom, P. (1995). High-Yield Noninvasive Human Genomic DNA Isolation Method for Genetic Studies in Geographically Dispersed Families and Populations. The American Society of Human Genetics.,1252-1254. doi:0002-9297/95/5705-0038$02.00

Mirmomeni, M., Ma, S. S., Sisakhtnez, S., & Doranegard, F. (2010). Comparison of the Three Methods for DNA Extraction from Paraffin-Embedded Tissues. Journal of Biological Sciences,10(3), 261-266. doi:10.3923/jbs.2010.261.266

Parzer, S., & Mannhalter, C. (1991). A rapid method for the isolation of genomic DNA from citrated whole blood. Biochemical Journal,273(1), 229-231. doi:10.1042/bj2730229

Pikor, L. A., Enfield, K. S., Cameron, H., & Lam, W. L. (2011). DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses. Journal of Visualized Experiments,(49). doi:10.3791/2763

Rohland, N., & Hofreiter, M. (2007). Comparison and optimization of ancient DNA extraction. BioTechniques,42(3), 343-352. doi:10.2144/000112383

Sengüven, B., Baris, E., Oygur, T., & Berktas, M. (2014). Comparison of Methods for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded Archival Tissues. International Journal of Medical Sciences,11(5), 494-499. doi:10.7150/ijms.8842

Shahriar, M., Haque, M. R., Kabir, S., Dewan, I., & Bhuyian, M. A. (2011). Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains. Stamford Journal of Pharmaceutical Sciences,4(1). doi:10.3329/sjps.v4i1.8867

Steinau, M., Patel, S. S., & Unger, E. R. (2011). Efficient DNA Extraction for HPV Genotyping in Formalin-Fixed, Paraffin-Embedded Tissues. The Journal of Molecular Diagnostics,13(4), 377-381. doi:10.1016/j.jmoldx.2011.03.007

Wilson, K. (2001). Preparation of Genomic DNA from Bacteria. Current Protocols in Molecular Biology,56(1). doi:10.1002/0471142727.mb0204s56

Zoetendal, E. G., Ben-Amor, K., Akkermans, A. D., Abee, T., & Vos, W. M. (2001). DNA Isolation Protocols Affect the Detection Limit of PCR Approaches of Bacteria in Samples from the HumanGastrointestinal Tract. Systematic and Applied Microbiology,24(3), 405-410. doi:10.1078/0723-2020-00060




    
              Karen Martin
GoldBio Marketing Coordinator


"To understand the universe is to understand math." My 8th grade
math teacher's quote meant nothing to me at the time. Then came
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along. But since math feels less tangible, I fell for biology and have
found pure happiness behind my desk at GoldBio, learning, writing
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Proteinase K Protocol Digestion Guide

This guide shows the experimental conditions for digestion with proteinase K.

Posted by Megan on August 1st, 2018  ⟩  0 comments

The traveling salesman, 45 and otherwise healthy, had been hospitalized several times already.

Based on a real homicide case.

The traveling salesman, 45 and otherwise healthy, had been hospitalized several times already. He complained of fatigue and weight loss, a condition that had persisted for half a year. Doctors hadn’t come to any diagnosis; they weren’t even sure if he had a disease, because no tests they’d conducted offered conclusive results. His main physician referred the salesman – we’ll call him Frank – to a pathologist, a doctor who often performs disease analysis in a forensic context. That pathologist, Dr. Daniel, asked for a sample from the patient that Frank’s physician may have thought unusual: fingernail clippings.

This story might be resemble the beginning of a medical or crime drama many of us are familiar with. However, much of the forensic science presented in crime shows is actually conducted in real labs. When a crime like robbery or murder is committed, scientists are consulted to help identify a perpetrator, and they often rely on DNA evidence from body fluids like blood. But if there is no biological evidence left behind by a criminal – as in the case of poisoning – forensics experts have alternate methods of discerning what substance may have been responsible.

This situation usually requires a toxicologist, an expert on chemical analysis and substance reactions, whose knowledge can identify what poisons are present in a body or tissue. The sources they collect may be blood or vitreous fluid from the eyes; they also sample nails or hair. Toxicological analysis can improve law enforcement’s chance of solving a crime if one exists, providing investigators with biochemical and DNA profiles. While Frank’s identity was known, his fingernail samples still carried supplementary evidence pivotal to diagnosing his mysterious ailment.

Dr. Daniel understood the forensic value inherit to this man’s nails and their cellular composition. As a medical professional, he knew the dynamics of tissue growth: cells rely on and grow in the matrix of the body, but they incorporate environmental chemicals and substances as well. These molecules accumulate in the cellular complex, and they can be stored in the body for years. Such chemicals can be detected much later through biochemical tests. Nails in particular have a slow rate of growth, offering retrospective insight to the past year’s growth depending on the individual and length of the nail. Substances can be detected, identified and quantified through techniques developed by forensic specialists.

Frank’s nail clippings were easily packaged and transferred for additional analysis; only a small portion would be necessary for the process. Though Dr. Daniel had already made a confident visual analysis on the nails, he wanted a second opinion, and the added confirmation would hold weight in a court system he was now certain the case was destined for. Ancillary tests had to be conducted by the toxicologist who next received Frank’s fingernail clippings along with the chain of custody, a document passed between investigators to conserve the legitimacy of evidence. The toxicologist signed the form and started planning the best tests for her examination of the nails.

Nails and hair have special advantages in the forensics laboratory. The process of collecting these samples is simple, noninvasive and negligible in the physical integrity of a victim or perpetrator. Both sources are also robust in their resistance to chemical change – the durability and slower decomposition rate of keratin preserves nails and hair, even at room temperature. There are specialized chemicals used to counteract these properties, and these would be employed by the toxicologist.

A study published in the Journal of Analytical Toxicology confirmed certain substances, incorporated into the keratin fibers themselves, can be detected in nails for 3–6 months. This study of over 10,000 samples also indicated nails provide a longer potential timeline of exposure than hair does, raising their value for the forensic scientist. The toxicologist had to consider the options of tests she would prioritize to develop her results and the most valuable conclusions. There were a few different theories she could make based on what tests she performed. Nails as a substrate can diagnose general or environmental pathologies, indicating health concerns like infectious disease or trace elements from chemical exposure.

Initial tests performed include enzyme-linked immunosorbent assay and liquid chromatography–tandem mass spectrometry (LC–MS-MS). Samples that test as presumptive positive then go through additional confirmatory tests. In preparation, they are washed, pulverized and chemically digested. This process is another familiar method to biochemical laboratories, employing many of the same techniques and materials. Depending on the extraction performed, tests can provide scientists with the sample’s DNA profile or a summary of incorporated chemical components.

The priority of this case, the extraction of unknown substances from biological samples, is an important duty of forensic specialists and medical examiners. Chemical extraction was performed by the toxicologist analyzing Frank’s nails. A similar process is used by forensic scientists conducting DNA identification using the extraction of exogenous DNA.

Protocol by the Office of the Chief Medical Examiner in New York City entails the processing of these samples, applied when nails contain DNA or chemical information to medicolegal cases. Their keratin composition is first degraded by a digestion buffer; organic extraction involves a digestion buffer of Tris (pH 7.5), EDTA, 0.1% SDS and proteinase K. The proteinase K is directly accountable for breaking down the tough keratin and thus freeing other molecules for inspection. Once the nails are digested, they’re incubated and centrifuged. Subsequently recovered substances (DNA, trace element, toxin or biochemical) are purified, concentrated and extracted for identification. Regardless of whether nail samples are processed individually or as a group, soaking clippings in digestion buffer results in the highest recovery rates of significant biomolecules.

Biochemical extraction is used in cases of assaults with direct contact between victim and assailant. DNA from the victim can be separated from that of the perpetrator for positive identification. It can also identify the person from whom the nail originated. One nail clipping constitutes a robust sample for DNA, and medical examiners have confirmed the sources can remain viable for upwards to twelve years after removal from live or deceased individuals. In a 2005 research project by medical examiners in Italy, nail samples from exhumed and skeletonized individuals (deceased within the last twelve years) were processed with another proteinase K, SDS and DTT buffer. The extracted DNA was then amplified with polymerase chain reaction to help produce a successful sequence. Their procedures yielded DNA sufficient to identify each individual when soft and bone tissue were inconclusive as compared sources, proving just one clipping can provide the identity of a person.

Just one clipping also – in Frank’s mystery illness – increased the toxicologist’s success rate for detecting other biochemical substances. She used the same technique a medical examiner might when isolating DNA, using a similar proteinase K buffer. Extraction was optimized by the forensic toxicologist to indicate and classify any toxins. This technique, usually used for drug detection, was recommended by Dr. Daniel to identify something more sinister: poisons.

The nails were analyzed for metals, including mercury, arsenic and lead; arsenic was detected in all samples. Dr. Daniel confirmed these findings with his own – multiple bands of transverse true leukonychia or “Mee’s lines.” These transverse white bands on the nails are a physiological presentation indicative of arsenic poisoning, and they were present on Frank’s nails upon Dr. Daniel’s initial visual analysis.

The diagnoses of both toxicologist and pathologist had an unpredicted result: Frank’s primary physician recalled a similar patient. Another traveling salesman, of the same age and sales territory, had also reported weakness and weight loss before dying of unexplained causes. The cases were too similar to ignore, and Dr. Daniel asked for a comparative study between the two salesmen. When the first individual was exhumed again, arsenic was also detected in his samples. Consequently, a criminal investigation was launched under suspicion of deliberate poisoning.

Similar cases of attempted and successful poisoning have occurred for centuries, with arsenic becoming less popular once detection tests were developed in the 1830s. They do, however, resurface occasionally, as in the 1995 murder attempted by St. Louis native Jim Boley against his wife, a case that garnered national coverage. More recently, a Chesterfield, Mo. case of domestic homicide involved mineral water laced with arsenic – refer to the baffling 2004 death of John Mullen. This later crime was months in the casebook before arsenic was identified. The forensic technique is more refined in today’s laboratories, with regulated procedures and quantities of reagents like the instrumental keratin-cutter, proteinase K.

Outside the lab, investigators discovered both salesmen (now regarded as victims of poisoning) regularly ate at the same department store lunch counter while they traveled. They were also consistently served by the same waitress. Based on the average daily growth rate of nails (0.1-0.15 mm), law enforcement approximated the dates during which the salesmen had consumed arsenic. The Mee’s lines of the undigested nail samples were measured, and an estimate of distance between incidents was made to prove multiple arsenic-related events. Based on the quantity of Mee’s lines formed, Frank’s exposure was substantial, perhaps over half a dozen meals laced with the poison. They aligned with logs from his traveling schedules; the Mee’s lines from both men corresponded to dates when they’d dined at the lunch counter. Investigators had new reason to pursue this waitress as a suspect.

In the interim of their analysis, Frank died of health complications directly associated to the arsenic poisoning. The case had once again ascended in criminal court, now to murder. With the laboratory’s instrumental results and testimony from Dr. Daniel, the waitress was arrested and convicted of murder. The conviction of the salesmen’s murderer prevented any more unknowing victims from dining on what would be their last meal. It also reassured the forensic community that nails are essential sources of evidence for such cases.

All this pseudo-noir drama and real-life crime, resolved with the help of a buffer that has been a useful tool in biochemical research.

References

Conklin, E. E. (2005, January 19). Dead Reckoning. Riverfront Times. Retrieved July 9, 2018, from https://www.riverfronttimes.com/stlouis/dead-recko...

Daniel III, C. R., Piraccini, B. M., & Tosti, A. (2004). The nail and hair in forensic science. Journal of American Academic Dermatology, 50(2), 258-261. doi:doi:10.1016/j.jaad.2003.06.008

Foran, D., Hebda, L., & Doran, A. (2015). Trace DNA from Fingernails: Increasing the Success Rate of Widely Collected Forensic Evidence (Rep. No. 249534). Retrieved July 9, 2018.

Hebda, L. M., Doran, A. E., & Foran, D. R. (2014). Collecting and Analyzing DNA Evidence from Fingernails: A Comparative Study. Journal of Forensic Sciences, 59(5), 1343-1350. doi:10.1111/1556-4029.12465

NYC Office of Chief Medical Examiner. (2016, October 14). Forensic Biology Protocols for Forensic STR Analysis [Digital protocol transcript]. Office of Chief Medical Examiner, New York City.

Piccinini, A., et. al. (2006). Forensic DNA typing of human nails at various stages of decomposition. International Congress Series, 1288, 586-588. doi:10.1016/j.ics.2005.08.029

Shu, I., Jones, J., Jones, M., Lewis, D., & Negrusz, A. (2015). Detection of Drugs in Nails: Three Year Experience. Journal of Analytical Toxicology, 39(8), 624-628. doi:10.1093/jat/bkv067


Megan Hardie is an undergraduate student at The Ohio State
University’s Honors Arts and Sciences program. Her eclectic
interests have led to a rather unwieldly degree title: BS in
Anthropological Sciences and BA English Creative Writing,
Forensics Minor. She aspires to a PhD in Forensic Anthropology
and MA in English. In her career, she endeavors to apply the
qualities of literature to the scientific mode and vice versa,
integrating analysis with artistic expression.
          Megan Hardie
      GoldBio Staff Writer
Posted by Fernanda on July 6th, 2018  ⟩  0 comments

For a few millennia, mankind has used honey as food and a healing agent without knowing how it worked. But, through experimentation, scientists have discovered that honey is a very complex substance. And today, we know that some of the components of honey that helps it be so effective at fighting disease are antibacterial factors.

Humans discovered the sweet powers of honey a few millennia ago. Paintings originating in the Middle Stone Age, 8,000 years ago, show humans collecting honey among bees. Romans ate honey to treat gut diseases. In ancient Egypt, honey was often used in combination with grease and lint to treat wounds. Nowadays, just like our ancestors, we continue to use honey to sweeten our food and treat various ailments. You might recall a time when your mom gave you some honey to soothe your sore throat.

In 1962, studies done by Jonathan White et al. revealed honey contains the enzyme glucose oxidase, which produces gluconic acid and the antimicrobial hydrogen peroxide, the antiseptic that doctors recommend for cuts and scrapes to inhibit bacterial growth. Yes! Honey can make hydrogen peroxide in the presence of a little water. In fact, honeybees add glucose oxidase to the collected nectar during honey production. This addition helps establish a low pH (3.5-4.5) and prevents bacterial growth, resulting in the preservation of honey.

It’s not just this one enzyme that gives honey its magic. A study done by Chirife et al. in 1983 suggests that the sugar in honey contributes to its antimicrobial activity. The supersaturation of honey with high sugar content means that there is very little water available for any organism to consume and thrive. Since bacteria need water to survive, the low amounts of water in honey makes it an effective antibacterial substance.

Not all honey is made the same, however. Not long ago, scientists found that Manuka honey, derived from the Manuka tree (Leptospermum scoparium) found in New Zealand, contains a very powerful antibacterial compound. In 2008, Elvira Mavric and her team studied 50 different samples of honey, including six New Zealand Manuka samples. They found that Manuka honey contained 100-fold higher amounts of the phytochemical methylglyoxal (MGO), compared to other honey. MGO is a powerful compound capable of killing cells that form from dihydroxyacetone (DHA) present in the nectar of L. scoparium flowers.

Manuka Trees near Queen Charlotte Sound, New Zealand

What’s more important about their findings is that unlike dilution of other types of honey, diluted Manuka honey effectively killed E. coli. MGO in honey is especially effective in inhibiting growth of S. aureus, the bacterium that can cause minor skin infections and serious diseases such as pneumonia and meningitis.

Pair the antimicrobial aspects of honey with technology, and researchers are able to produce medical-grade honey, used specifically for wound care. This type of honey is generated by sterilizing raw honey with gamma irradiation to destroy any bacterial spores that if left in the honey, may cause wound botulism or gangrene. Nowadays, the main medical-grade honeys used are Manuka honey and Revamil, a honey produced through a standard process in greenhouses.

Medical-grade honey has also been very useful in uncovering the factors that make honey a potent killer of bacteria. Paulus Kwakman and his team studied the “unknown bactericidal factors” in unprocessed Revamil honey. They found that unlike Manuka honey, Revamil contains the antimicrobial peptide (AMP) bee defensin-1, also called royalisin. Bee defensin-1 was first identified in honeybee blood (hemolymph) as food for queen bee larvae. Kwakman et al. showed bee defensin-1 in honey has strong antibacterial activity against Gram-positive bacteria including B. subtilis, methicillin-resistant S. aureus, and Paenibacillus larvae, which causes a devastating bee larval disease. And, just last year, Marcela Bucekova and her research team published their studies showing how bee defensin-1 stimulated wound closure in vitro and promoted re-epithelisation in uninfected excision wounds in rats.

Now that we live in an age when organisms are becoming resistant to commonplace antibiotics and emerging “superbugs,” the medical and scientific communities are recognizing the healing potential of honey. For example, S. aureus is one bacterium that people carry, and it can cause many diseases. In the mid-twentieth century, however, S. aureus became resistant to methicillin, an antibiotic close to penicillin, giving rise to the superbug MRSA. We know that MRSA is found in (colonizes) the nose tissue of about two in 100 individuals, where it usually does not cause disease. But, if the colonized skin is injured, MRSA could enter the body and cause an infection leading to devastating skin destruction and death. This is why MRSA is called “flesh-eating bacterium.”

Actually, Toney Poovelikunnel and his team discovered very recently that medical-grade honey is able to eliminate (decolonize) MRSA from the nose of patients. In this clinical study, Poovelikunnel and his team applied medical-grade honey to the anterior nares (the external part of the nostrils) with a cotton swab, three times a day for five consecutive days. They learned that, medical-grade honey has a decolonization rate of 42.8%, similar to that of mupirocin, a topical agent frequently used to decolonize MRSA from the anterior nares. Who would have thought, honey can fight the dreaded MRSA superbug?

These studies certainly indicate that honey can help us fight antibiotic resistance. And, while we continue to discover new ways in which honey can combat dangerous bacteria, we now have a better understanding of the mechanisms behind honey’s healing powers.

References

Boukraâ, L. (2013). Honey in traditional and modern medicine. Boca Raton, FL: CRC Press. Doi:10.1201/b15608.

Bucekova, M., Sojka, M., Valachova, I., Martinotti, S., Ranzato, E., Szep, Z., . . . Majtan, J. (2017). Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo. Scientific Reports, 7(1). Doi:10.1038/s41598-017-07494-0.

Cooper, R., Molan, P., & Harding, K. (2002). The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds. Journal of Applied Microbiology, 93(5), 857-863. Doi:10.1046/j.1365-2672.2002.01761.x.

Dams, L. R. (1978). Bees and Honey-Hunting Scenes in the Mesolithic Rock Art of Eastern Spain. Bee World, 59(2), 45-53. Doi:10.1080/0005772x.1978.11097692.

Ghalioungui, P. (1987). The Ebers papyrus: A new English translation, commentaries and glossaries. Cairo: Academy of Scientific Research and Technology.

Henriques, A. F., Jenkins, R. E., Burton, N. F., & Cooper, R. A. (2010). The effect of Manuka honey on the structure of Pseudomonas aeruginosa. European Journal of Clinical Microbiology & Infectious Diseases, 30(2), 167-171. Doi:10.1007/s10096-010-1065-1.

Kwakman, P., Akker, J. V., Güçlü, A., Aslami, H., Binnekade, J., Boer, L. D., . . . Zaat, S. (2008). Medical‐Grade Honey Kills Antibiotic‐Resistant Bacteria In Vitro and Eradicates Skin Colonization. Clinical Infectious Diseases, 46(11), 1677-1682. Doi:10.1086/587892.

Kwakman, P. H., Velde, A. A., Boer, L. D., Speijer, D., Vandenbroucke-Grauls, C. M., & Zaat, S. A. (2010). How honey kills bacteria. The FASEB Journal, 24(7), 2576-2582. Doi:10.1096/fj.09-150789.

Kwakman, P. H., & Zaat, S. A. (2011). Antibacterial components of honey. IUBMB Life, 64(1), 48-55. Doi:10.1002/iub.578.

Mavric, E., Wittmann, S., Barth, G., & Henle, T. (2008). Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand. Molecular Nutrition & Food Research, 52(4), 483-489. Doi:10.1002/mnfr.200700282.

Poovelikunnel, T., Gethin, G., Solanki, D., Mcfadden, E., Codd, M., & Humphreys, H. (2018). Randomized controlled trial of honey versus mupirocin to decolonize patients with nasal colonization of methicillin-resistant Staphylococcus aureus. Journal of Hospital Infection, 98(2), 141-148. Doi:10.1016/j.jhin.2017.10.016.

White, J. W., Subers, M. H., & Schepartz, A. I. (1963). The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochimica Et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects, 73(1), 57-70. Doi:10.1016/0926-6569(63)90108-1.


Categories: 79101

Posted by Chris on June 14th, 2018  ⟩  0 comments

It’s summer time again! The temperature has hit “beautiful”, the trees and grass have turned back to their vibrant shades of green, and the birds have begun their annual serenades. Ah, sweet wonderful summer.

Except that we’re all stuck in the same old labs

staring at the same instruments

the same shelves full of bottles and tubes

the same old posters on the walls that we’ve been staring at all through the days of winter

It’s enough to make a scientist go mad! Mad, I say!

But before you let that happen, just go out for a short walk outside (or a long walk, depending on how close you were to mania) and take a deep breath of the sweet, pollen heavy summer air (unless you’re one of the 20 million adults who are hyper-sensitive to the pollen) and relax. We’ve got some fun, low stress, summer-friendly experiments for you to keep your experimental interests fresh without all the stress of those research deadlines.


DIY Earthworm Jar

Have you ever wondered what earthworms actually do in the ground? Worms are some of our best soil “scientists”. They help aerate the soil, break down organic matter and help to turn the soil over in order to mix the layers to provide better plant nutrition. According to the University of Illinois, 500,000 worms on an acre of land will make 50 tons of fertilizer casings and can create a drainage system equal to 2000 feet of 6 inch pipe! This little experiment can help illustrate exactly how worms mix the soil layers.

Materials

Earthworm

  • 2-6 earthworms
  • From the yard: After a rainstorm many will come up to the surface to breathe. Also if there is a part of your garden with good rich soil there are likely worms there that should not be too hard to dig up.
  • From the store: Most bait shops sell both night crawlers and standard earthworms. Some gardening centers sell Red Wrigglers for composting.
  • Two liter soda bottle or similarly sized jar or container (or an ant farm type container might also be good!)
  • Rich Soil – either from the garden or a topsoil mix
  • Sand (not chemically treated) – lighter will show up better
  • Coffee Grounds, Veggie/Fruit Scraps – although stay away from more acidic foods like onions, tomatoes and citrus.

Optional:

  • Cheesecloth and Rubber band or lid with holes for ventilation
  • Thick paper or cloth to cover the sides of the habitat and keep out light
  • Notebook for observations/ drawings
  • Measuring tape/kitchen scale
  • Wire mesh screen/colander
  • Soil Thermometer/Soil pH meter

Prepare Container

  1. Wash out container making sure it’s completely rinsed out with no soap residue.If you’re using an empty two liter cut the top of the bottle off where it starts to slope.
  2. If your container has a lid, poke holes in the top to let in air.
  3. Fill the jar with a layer of sand (½ inch) and soil (2 inches). Make sure you wet the soil and sand so that they are damp but not soaking.Continue layering sand and soil until the jar is full, your last layer should be a soil layer. Make sure to leave an open space of at least 2 inches at the top.

Add worms

  1. Before you put the worms in, count and weigh the worms and measure their average length so you can keep track of their growth.
  2. Put coffee grounds and food scraps on top of the soil.
  3. Add a lid with holes on or cover the top with cheese cloth and secure with a rubber band in order to help keep bugs out.

Keep the worms happy

  1. Worms don’t like light, so either wrap a piece of dark paper around the jar or store the project in a cool, dark place.
  2. Check on the worms every few days to make sure that the soil is still moist.
  3. Give them new food about once a week.
  4. Pretty soon you should be able to see the layers of sand and soil start to mix together and tunnels from the worms traveling through the soil.

Optional Science (for the overachievers)

  • Compare the ambient temperature to the temperature of the soil.
  • Compare the soil pH across the duration of the experiment to see how it has changed.
  • Measure and track the soil migration based on worm number or initial size.

After a few weeks of observing and keeping the worms it’s time to let them go back out into the wild. Use a wire mesh screen to help separate the worms from the dirt.Weigh and measure the worms and compare this to your original measurements and bid them a fond adieu!

References
http://extension.illinois.edu/worms/live/
http://wemadethat.blogspot.com/2013/04/diy-earthworm-habitat.html?m=1
http://www.bbc.co.uk/gardening/gardening_with_children/homegrownprojects_watchworms.shtml#tips_and_advice


Backyard Birding

Birds are the trumpeters of spring and work as one of our greatest natural pest control. To a scientist, they can also be a wonderful subject of exploring animal behavior and genetics. Backyard birding is the idea of not only observing the birds in your area, but also putting things in place to allow them to thrive.

The best resource to explore is the National Audubon Society’s website for tips. There are resources to help you identify different birds through sight or through their calls. They also have guides on different types of feeders and housing you can make. Different seed and nest boxes can attract many different types of birds. You can also buy some great bird observing window feeders, like this one (not sponsored), which offers a 2-way mirror system so that you can watch birds up close at your window without scaring them off. One issue with this bird feeder, however, is that due to the size of a window, all the uncovered area still exposes you and scares the birds away. A way around that is to use Glad Press’n’Seal (or similar) around the other open areas of the window.

Spring is usually the best time to start your observations, with early mornings and late evenings being a great time to watch birds in your yard. But you can also help create a perfect environment for a range of seasonal birds to thrive in your area all year long.


Home Sweet Biome (Biomes in a Bag)

Have you ever wondered exactly how your environment affected the rate of plant growth in your area? Science Buddies has a great experiment procedure to figure that out exactly! This is a quick and simple science experiment that is enlightening and needs very little upkeep.

Materials

  • 2-liter plastic bottles (6), clean and empty
  • Bag of river rock
  • Bag of potting soil
  • Packets of the same type of seeds; vegetables such as beans or peas work well, but flower and grass seeds work, too (need about 20 seeds, total)
  • Resealable food storage bags, 1 gallon-size (6)

Procedure

  1. To model two terrestrial biomes, the temperate forest and the tropical forest, find a place that is sunny (like a sunny windowsill) and another spot that is shady and a bit warmer than the first location.
  2. Cut each of the plastic bottles in half.
  3. Place a handful of river rocks in each plastic half. The rock layer should be 1 inch deep.
  4. Put a layer of potting soil on top of the rocks in all six bottles. There should be twice as much soil as there are river rocks, so approximately 2 inches.
  5. Now, drag your finger in the middle of the soil and make a trench as deep as your fingernail.
  6. Place 3-4 seeds in the trench and then gently replace the soil over the seeds. Spread the seeds out—don't put them all in one pile together. Repeat steps 5-6 for the other five containers.
  7. Fill the measuring cup with water and carefully water one container at a time until you see the water collect at the bottom of the rocks. The amount of water you'll use for each container should be between ¼ cup and ½ cup. The soil should not be soaking wet or soupy, just moist. Make sure that the seeds remain covered after you have watered. Repeat this step for all of the containers.
  8. Now place each container in a resealable plastic bag. Label each bag with the amount of water in that container and the location where it will be placed. Three containers should be in one location and the other three should be at the other location. Carefully and completely seal each plastic bag.

That’s it! You won't have to water the seeds again, because the water in the bag recycles itself! The roots of the plant absorb the water, which then travels up the stem to all the parts of the plant. Some of the water in the leaves evaporates, as does some of the water in the soil. The evaporated water condenses on the inside of the bag and forms water drops. Some of the water drops fall back into the container like rain. Once the water falls back into the plant container, the water cycle starts again.

Observe the biomes twice daily for the next week. Once in the morning and then again in the evening. Try to observe the biomes at the same times each day. Have any seedlings come up? How long did it take for seedlings to come up? Use a ruler to measure how tall the seedlings are. Has water condensed on the inside of the bag? How moist does the soil look?

Reference
Science Buddies Staff. (2017, July 28). Home Sweet Biome: How Do Plants Grow in Different Environments? Retrieved from https://www.sciencebuddies.org/science-fair-projects/project-ideas/EnvSci_p046/environmental-science/biomes


Pollinator Garden

Pollinators are an important part of the ecosystem and a lot of our heavy lifters such as the honeybee and monarch are having a hard time. Help them along this spring by creating a pollinator garden!

One of the biggest ways to really attract pollinators to your hard is to plant milkweed. There is a great video series on Youtube about how to find, germinate and plant milkweed. The first video of the series also lists a few places where you can get free milkweed seeds. Just be careful to manage milkweed because it can get a little out of hand and overrun other bushes and trees. Another great resource for tips on growing a pollinator garden can be found on the USDA’s Forest Service page.

May/June is a great time to get started! Get those seeds and start germinating. While you wait on the little sprouts, carve off some area to dedicate to the garden.


Regrow Celery (Great to do with kids!)

Rather than immediately scrapping or composting that old celery stock, why not save it for regrowth? Children will not only observe plant growth over time, but will also learn an important lesson in sustainability and science.

The best website with information on how to regrow celery from stalk is found here. This page not only shows the initial kitchen growing techniques, but also goes into detail about when to transplant it outside.

Transplanting time will depend on your location. Celery does best in cool weather when nighttime temperatures are around 55°F (13°C) and average daytime temperatures stay between 60-70°F (16-21°C). For more information on optimal, outdoor growing conditions, check out the Aggie Horticulture website.


Water Cycle in a Bag (Great to do with kids!)

Teach your kids about the water cycle in a very fun, hands-on way with this Water Cycle in a Bag activity found on playdoughtoplato’s website. According to the page, it’s quick to prepare and engages kids for a few days.

The instructions on how to set this up is featured on the website. But the basic idea is this: you get a zip up sandwich bag, draw a cloud and sun up top. Fill the bag with just a little water and blue food coloring. Seal it up and tape it to the window. Check it with the kids each day to watch the levels evaporate, condense and trickle down.

This is a great activity when you’re going to have lots of sun. And with school ending soon, late spring and early summer will be a fun time for the kids to do this activity.

And finally...


M&M Hunter Challenge

Camouflage is one of nature’s best defenses for prey species. For this delightful and delicious little “lab” experiment, you might need to get some help from your lab mates. Begin by mixing certain colored M&M’s in a Skittle-based habitat to see how camouflage can help to keep M&M’s “alive” against the hungry lab predators in a set amount of time! You might have to promise to let your lab mates keep any M&M’s they “capture”, but not before you count how well they did. Make it a party and see which researcher is the ultimate M&M predator.

Materials

  • Plastic baggies (6)
  • M&M's, at least 10 of each color
  • Use plain M&M's, which should have six colors: Yellow, blue, green, brown, red, and orange.
  • To make sure you have at least 10 candies of each color, you will want to get at least two 1.69-oz. packages.
  • Skittles®, at least 60 of each color
  • Use plain Skittles, which should have five colors: orange, yellow, green, red, and purple.
  • To make sure you have at least 60 candies of each color, you will want to get at least one 16-oz. package.
  • Metal pie tin or sturdy paper plate
  • Stopwatch or timer
  • 2-4 volunteer predators who like to eat M&M's

Procedure

  • First you will need to prepare a mixed group of "prey." Do this by counting and placing 10 M&M's of each color into a plastic bag.
    • This means you should have one plastic bag with 10 yellow, 10 blue, 10 green, 10 brown, 10 red, and 10 orange M&M's candies in it.
  • Prepare different "habitats" using Skittles candies. Do this by counting and placing 60 Skittles of a single color in a bag. Repeat for each color, in the end you will have 5 bags — each with just one color of Skittles.
    • This means you should have one plastic bag with 60 orange Skittles, one bag with 60 yellow Skittles, one with 60 green Skittles, one with 60 red Skittles, and one with 60 purple Skittles.
  • Gather together your lab of 2-4 volunteer "predators." This can be anybody who likes to eat M&M's.
  • Explain the rules of the game to your predators as follows:
    • The volunteers should pretend to be M&M's birds. They should make a "beak" using their pointer finger and thumb for collecting M&M's candies.
    • You will set a timer (or watch a stopwatch) for 20 seconds. During those 20 seconds, the volunteers will use their beak to quickly pick up M&M's and quickly put them in their other hand.
      • To encourage the volunteers to be fast, tell them that when they are done with the experiment, they can eat the same number of candies as they picked up. (But they should not eat any candies until you are all done testing.)
    • The volunteers should avoid picking up any Skittles candies because Skittles make the M&M's birds sick. The Skittles represent the habitat that the M&M's candies live in.
  • After explaining the rules, pour one prepared bag of Skittles into a metal pie tin or sturdy plate. Mix in the prepared bag of M&M's. Put the pie tin in the middle of your group of M&M's birds. Make sure everyone can reach the pie tin.
  • Set your timer for 20 seconds.
  • Say, Go! And start the timer. When the timer beeps, everyone should stop picking up M&M's.
  • Count the number of each M&M's color that each person collected If any volunteer collected any Skittles, put the number of Skittles they collected in the bottom row of their column, the one labeled "Skittles." Also, re-emphasize that Skittles make the M&M's birds sick and should be avoided.
  • Enjoy your treats!
Posted by unknown on June 7th, 2018  ⟩  0 comments

      Leaving your things lying around is something parents have lectured about forever, but the rule of tidiness is even more important in a laboratory setting. Biochemical products are a bit more volatile than dirty laundry, so it's important to have all of your chemicals accounted for. This article reviews some of the frequently used substances which may be left unattended and concern technicians – are they still functional, or are they damaged beyond repair? It's necessary to understand which substances are sensitive to neglect, but we hope to assuage some unwarranted panic over robust materials.

      Left products on the lab bench - troubleshooting

      What we'll talk about:

      1. Antibodies
      2. Enzymes
      3. DNA
      4. PCR-amplified samples
      5. BSA
      6. ELISA
      7. Bacterial cultures
      8. Slides
      9. Hygroscopic materials
      10. Frozen substances


      Before proceeding, I want to say that this article is intended to be more informative about products that have been accidentally left out. It is not guaranteed that all reagents will behave the same way and other factors can influence performance. Therefore, do not be careless with products in your lab and always store them according to manufacturer or supplier instruction



      1.   Antibodies:

      • The product: Antibodies have variable shelf-life depending on type, but they’re generally kept with glycerol or sucrose to prevent aggregation and promote functional longevity. While shelf-life can differ, many kept at ideal conditions: temperature, pH, desiccation and lighting specifications recommended by the manufacturer can remain functional for more than a year.
      • The situation: You’ve returned to the lab after a weekend and realize you left an entire box of antibodies on the counter, sitting at room temperature. The instructions inform you that storage environment should be 4°C.
      • The diagnosis: There's still hope. Don't throw them out before you test them. Suppliers have done experiments on the storage and shipment of antibodies at various temperatures, and leaving them at ambient temperature even for a week did not decrease their effectiveness. Some companies even subject their antibodies to 37°C and higher temperatures for performance quality testing.
      • The precautions: Purified antibodies have characteristic stability, but it has never hurt anyone to be cautious and run validation tests to be sure.


      2.   Enzymes:

      • The product: Storing enzymes usually involves -20°C temperatures and the addition of glycerol to prevent protein denaturation. Their use in experiments is sometimes cumbersome, because it’s recommended they be kept frozen in the process.
      • The situation: After conducting a reaction, another technician points out your restriction enzyme, no longer in the cooler you used to transport it from the fridge.
      • The diagnosis: Enzymes usually won’t be destroyed by a couple of hours outside a freezer, and they’ll survive a power outage too. A test on a group of 23 unmodified restriction enzymes stored and shipped at ambient temperatures revealed they can remain active without being refrigerated for one to three weeks. We won’t recommend leaving them on the bench for nearly a month, though.
      • The precautions: Enzyme integrity is damaged by temperature fluctuations, so thawing and refreezing products too many times will cause denaturation. Even leaving enzymes in the door of a freezer can be risky due to unmediated temperatures. Avoid protease contamination when enzymes are in open environments.



      3.   DNA:

      • The product: DNA is incredibly complexand has to be treated with care to prevent contamination and denaturation. This is commonly done by keeping it isolated in temperatures of -20°C or lower.
      • The situation: You’ve been using DNA samples since 7:00am. At 5:00pm, you’re struck with the thought that with every minute you’ve had the DNA sample out, the strands have slowly been breaking apart, invalidating the ten hours of work you just put in.
      • The diagnosis: DNA stored in a dry room temperature environment will degrade, but the speed of this process is not as rapid as you might think. Long-term storage can be conducted anywhere between -80°C and 4°C, but room temperature is safe for short-term as long as contamination is not a concern with the addition of EDTA and Tris. Stability is maintained longer by buffers.
      • The precautions: The freezing/thawing process, if repeated often, can damage DNA just as badly as leaving it in unmediated temperatures. Regardless of storage temperature, samples should be tested for concentration and evaporation. 


      4.   PCR:

      • The product: Related to DNA, polymerase chain reaction (PCR) techniques involve mixing DNA with polymerases, primers and other essential compounds to amplify DNA strands. Scientists commonly transfer PCR samples directly to 4°C refrigerators after the thermal cycling process.
      • The situation: You start your PCR and walk away. The last cycle finishes two minutes after you leave for your one-hour lunch break. When you come back to the machine, you see that it’s been waiting for extraction at room temperature.
      • The diagnosis: Inside of a PCR tube, DNA samples can retain their stability. Successful amplification will allow them to resist ambient environments for weeks, if not longer, without noticeable degradation. PCR itself is used to study ancient DNA samples left exposed to the elements for centuries; if that sample can still generate results, so can the one you left on the counter. It's important to note that many thermal cyclers will keep the tubes cool after the cycling process. Even if the machine were somehow unplugged or turned off, your samples should be OK.
      • The precautions: PCR machines must be given the same attention you intend for your samples. If left cooling overnight, the machine's overall lifetime could be impacted. A less reliable device will produce less reliable amplification. 

      5.   BSA:

      • The product: Bovine serum albumin (BSA) is a blocking agent commonly used in experiments detecting the presence and relationships of protein. It is rendered from bovine blood serum, reducing residual binding capacity and nonspecific interactions.
      • The situation: While conducting an ELISA experiment, you had a good number of tubes out. Your dried BSA was in a particularly small tube, and sometime in the process of things it was misplaced among the others you’d finished using. When you clean the bench at the day’s end, you find the unfinished BSA still among the empty bottles.
      • The diagnosis: BSA, especially when dried and used for blocking, will be fine for days without refrigeration. Powders need to be kept dry more than cold. Powder and stock solutions should also be sturdy for a few days if they’re misplaced over the weekend.
      • The precautions: Contamination from fluids is more of a problem than heat with this chemical, so it will be more of a concern if the left out container was also open.


      6.   ELISA:

      • The product: An enzyme-linked immunosorbent assay – ELISA – tests for substances in biochemical fluids via an assay of antibodies and color. Antigens are detected in samples by specifically-targeting antibodies, and serum (like BSA) is commonly screened through ELISA kits.
      • The situation: Testing for the viral antigen in a sample you’ve created, you put together an ELISA kit and go through the complex-forming process. The lab manager calls you away to perform some “housekeeping” tasks. Two days later, you remember your ELISA test.
      • The diagnosis: Whether left on the bench or put in the wrong freezer, ELISA tests are fairly hard to damage in short periods of time. They can sit at ambient temperature without the results degrading, and a kit that has been unintentionally frozen will thaw out with minimal damage.
      • The precautions: Extended periods of time can decrease activity in the test, but results should still be reliable. Avoiding contamination as an overall standard procedure is also important when a kit, used or otherwise, is left unattended.


      7.   Bacterial cultures:

      • The product: Bacteria are finicky; too much heat or cold will degrade their value as scientific subjects, because temperature fluctuations damage cells. The necessary storage conditions are highly dependent on strain, but refrigeration is generally suggested. Frequently used samples are ideally stored at 4°C while those kept for longer periods will be frozen or freeze-dried.
      • The situation: Your bacterial cultures are just starting to show promise in the last minutes of a Friday lab session. You consider leaving them for the weekend rather than making a fresh grouping Monday. There are risks, right? Mutations, contaminations, etc. Your intern says he once left a sample out for a week and came back to find half a dozen strains in one petri dish (though considering the state of his work bench, you aren’t surprised).
      • The diagnosis: Though there are sometimes recommendations to harvest culture DNA or plasmid quite quickly (within 12-24 hours), bacterial strains have been stored for months and years in laboratory settings without adverse effects on their utility. Random mutations will take more than a weekend to transpire. The real concern is contamination or competitive growth in the media if it’s fully exposed to the environment.
      • The precautions: Plasmid yields are more promising in fresher samples. Some strains are more sensitive and conducive to recombination than others, so unwanted developments are more likely to occur with a long, unmediated incubation period. It also isn’t out of the question that a large quantity of pelleted strains, left out for a weekend, will cause quite a smell.


      8.   Slides:

      • The product: Microscopic slides are inseparable from techniques in immunology, cellular cultures and organism selection. Microscopy is associated with slide production for viewing single-celled organisms and other small (but microscopically visible) results of experimentation. Slides themselves are fragile, and sometimes so are the samples they contain.
      • The situation: You’re a technician assigned to the care of several dozen slides from a friend’s month-long cellular immunology project. You leave the samples on a shelf for the week your colleague is on vacation; needless to say their return initiates a rather awkward conversation involving laboratory protocol and sample storage. Was their research ruined?
      • The diagnosis: Slides with properly fixated, embedded and sealed samples can be kept at room temperature as long as the ambient environment is standardly clean and free of agents like persistent mold.
      • The precautions: If the container was left open and exposed to light, this could be more of a problem. Artificial and natural light at high concentrations can harm slide integrity. Compare controls to see if anything has deteriorated.


      9.   Hygroscopic materials:

      • The product: Hygroscopic compounds are defined as such for their water-attracting characteristics. They are able to absorb and hold water molecules from the air. It's important to keep them in desiccated states or in the presence of desiccants.
      • The situation: Your dimethyl sulfoxide (DMSO) has been out in the lab for a good couple of weeks before you notice it sitting on the table. It’s conveniently placed about two feet from the laboratory sink.
      • The diagnosis: Hygroscopic compounds, if encountering high amounts of moisture in an improperly sealed container, are going to be damaged. Even in a properly desiccated environment, leaving these substances for too long without changing desiccants will expose them to excessive moisture. Being attentive to indicators is important, and if they signal compromised material, your product probably isn’t usable anymore.
      • The precautions: Desiccated materials can very sensitive. They require special protection from air, not because of temperature, but because of moisture, and this makes it harder to distinguish good from bad until they're quality-tested.


      10.   Frozen substances:

      • The product: Biochemical samples are almost universally kept cool. Frozen substances are further defended from heat-provoked transformation; they’re thawed before use and returned to consistently low temperatures. This category ranges from bacterial strains to pure compounds.
      • The situation: You have a stock of bacterial plasmids frozen for communal use in the lab. In the course of a day, you’ve removed the stock from the freezer, placed it on the bench, realized you needed to reorganize the entire freezer and come back to the stock seven hours later to find it in liquid form. Are the other techs going to ostracize you?
      • The diagnosis: There is no one answer here. There are circumstances when a chemical or sample is frozen to prevent degeneration, and allowing it to thaw out and maintain an ambient temperature will destroy it. Alternatively, recoveries of frozen E. coli strains after hurricane-produced power outages have been up to 94% successful even after over a month. Your bacterial plasmids should also be fine. Cases must be judged individually based on the substance’s function and traits.
      • The precautions: As previously stated, some chemicals will only function for as long as they are protected from temperature fluctuations. Check the labels to see how they're meant to be stored, and always be aware of your freezer settings.


      A concluding thought: vigilance is essential for chemical preservation and accurate science, but it’s equally as important to consider the durability of accidentally neglected materials. Don’t rush in your panic to discard all evidence of a wandering scientific mind – if you have safe methods, good controls and experience with the substance, you should be able to identify if something is still operable versus when it belongs in the trash.



      Resources

      Best long term storage method for DNA? (2013, July). Retrieved June 28, 2017, from https://www.researchgate.net.

      Clark, J., March, J. B., & Mdegela, R. H. (2000). Extended stability of restriction enzymes at ambient temperatures. Biotechniques, 29(3), 536-542. Retrieved June 19, 2017, from https://www.ncbi.nlm.nih.gov/pubmed.

      Cody, W. L., et al. (2008). Skim Milk Enhances the Preservation of Thawed -80°C Bacterial Stocks. Journal of Microbiological Methods, 75(1), 135-138. doi:10.1016/j.mimet.2008.05.006.

      Johnson, M. (2012). Antibody Shelf Life: How to Store Antibodies. Materials and Methods, 2(120). doi://dx.doi.org/10.13070/mm.en.2.120.

      MiniPCR. Debunking the 4 degree myth: PCR can be left at room temperature overnight. (2016, December 2). Retrieved June 28, 2017, from  http://www.minipcr.com/classroom-tips.

      Moran, E. (2013, May). Using Enzymes at the Bench — Keep it in the cooler? On ice? or at RT? Retrieved June 19, 2017, from http://bitesizebio.com.

      Wu, J., Kim, L., T. K., Huang, C., & Anakella, B. (2009, May). Stability of Genomic DNA at Various Storage Conditions [Scholarly project]. In SeraCare Life Sciences. Retrieved June 19, 2017, from http://www.colorado.edu.



                   Megan Hardie
               GoldBio Staff Writer

      Megan Hardie is an undergraduate student at The Ohio State
      University’s Honors Arts and Sciences program. Her eclectic
      interests have led to a rather unwieldly degree title: BS in
      Anthropological Sciences and BA English Creative Writing,
      Forensics Minor. She aspires to a PhD in Forensic Anthropology
      and MA in English. In her career, she endeavors to apply the
      qualities of literature to the scientific mode and vice versa,
      integrating analysis with artistic expression.

      Category Code: 79104, 88221, 79109, 88251