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For this exercise, you will perform a filter paper disk diffusion assay to determine the effectiveness of 5 different types of disinfectants.
Materials: 1 TSA plate/student, liquid cultures of Staphylococcus aureus and Escherichia coli, sterile cotton swabs, filter paper disks, 5 different disinfectants, forceps, and beakers with 70% ethanol
Go up to the instructor’s table and write down the names of the 5 disinfectants you will test in the chart below.
|Disinfectant #||Name||Concentration||Active ingredient/class of disinfectant|
|6||Sterile distilled water (control)|
1. Choose one of the bacterial species listed above. Use a cotton swab to inoculate a lawn of bacteria on your TSA plate. Discard swabs in the beaker provided at your table.
2. Label your TSA plate with the name of the microbe you are using, and numbers 1-6 evenly spread out on the plate (see demo plate done by your instructor). The numbers (and corresponding disks) should not be too close to the edges of the plate.
3. Bring your inoculated plate up to the front table, where you will be adding filter paper disks soaked in disinfectants.
4. Before each use, forceps should be flame sterilized as follows:
a) Remove forceps from beaker of alcohol. Keep the tips angled down at all times.
b) Put the forceps in the Bunsen burner flame just to ignite the alcohol.
You are not heating the forceps, just igniting the alcohol.
c) Keep a careful eye on the forceps (hold them steady in one location) until all the alcohol has burned off (this will be very quick). To avoid fires, do not hold them over any beakers of alcohol!
d) Once the alcohol has burned off, use the forceps to pick up one filter paper disk from the glass Petri dish.
5. Dip the disk into disinfectant #1.
Just touch the surface of the liquid—the disinfectant will soak into the disk by capillary action. It is important to not have the disks too wet when you place them on your TSA plate.
6. Place disinfectant #1 on the appropriate area of the agar plate. Tap it down gently with the forceps to ensure that it adheres to the surface of the agar.
7. Repeat this step for all disinfectants.
If you want to test some other product, just omit one of the disinfectants in the front of the room.
8. Add a filter paper disk that has been dipped in sterile water to the area of the plate labeled “#6”- this will serve as your negative control.
9. After each use, place the forceps back into the beaker with 70% ethanol. DO NOT heat them after use before returning them to the beakers.
10. Incubate plates (inverted, as usual) until the next lab period.
B. Antibiotic susceptibility testing
Each table: 3 large (150 mm) Mueller-Hinton agar plates, liquid cultures of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, sterile cotton swabs, antibiotic disk dispensers.
1. Work as a table for this exercise. Use a sterile cotton swab to inoculate the Mueller-Hinton agar plates with a lawn of bacteria for each species listed (one species/plate). Be sure to cover the entire area of the plate. This can best be accomplished by swabbing the entire surface of the plate three times, rotating the plate approximately 60º each time, and finishing the swabbing by going around the entire outer surface of the plate.
2. After all plates are inoculated, your instructor will show you how to use the disk dispenser to introduce antibiotic disks onto the plates. Be careful to always keep the dispensers in the upright position.
a. You will notice that each disk that is introduced to the plate is stamped with a letter and a number—these indicate the type of antibiotic as well as the concentration. For example, P10 is an abbreviation for 10 units of penicillin; PIP-100 is an abbreviation for 100 mcg (micrograms) of piperacillin.
3. Return to your lab table, and use your inoculation loop or needle to gently push down on the disks to ensure that they are adhered to the agar surface.
Flame your loop in between plates to avoid cross-contamination
4. Incubate the Mueller-Hinton agar plates (inverted) until the next lab period.
C. Demonstrating antibiotic production with Streptomyces
Materials: Soy Flour Mannitol (SFM) plates containing cultures of Streptomyces coelicolor and two additional Streptomyces cultures, liquid cultures of Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis, TSA agar plates (1/pair of students), sterile cotton swabs, sterile 1000 µl pipet tips.
Before beginning this experiment, write down the names of the organisms you will be testing here, and provide a brief description of their appearance.
|Strain #||Strain name||Description||Additional information|
1. Divide your plate into 4 areas (labeled 1, 2, 3 and – for negative control).
2. Choose one bacterial liquid culture from the list above, and use a sterile cotton swab to inoculate a lawn of bacteria. Discard swab in the beaker at your table.
3. Using the large round end of a sterile pipet tip, punch a hole out of the center of each of the 4 quadrants you created on your TSA Plate. Discard the tip in the disinfectant beaker.
4. Use another sterile tip to cut out a plug of agar from the lawn of the corresponding SFM plate (your instructor may provide these for you).
5. Transfer this plug of agar into the well you created in your agar plate.
6. Repeat for the other two Streptomyces strains, using a fresh sterile tip each time.
7. Place a plug of uninoculated SFM agar on your plate in the negative control quadrant.
8. Incubate the plates at 28˚C until the next lab period.
Management procedures in a fishery based on highly variable stocks and with conflicting objectives: experiences in the South African pelagic fishery
The pelagic fishery in South Africa targets mainly anchovy, Engraulis capensis, and sardine, Sardinops sagax, both of which have varied substantially in abundance during the history of the fishery. Since 1988, there has been progress in this fishery towards the use of management procedures as the basis for determination of management regulations, where a management procedure is defined as a set of rules, derived by simulation and normally implemented for three to five years, specifying how the regulatory mechanism is set, the data collected for this purpose and how these data are to be analysed and used. Advantages of management procedures include formal consideration of uncertainty, the ability to choose decision rules based on their predicted medium-term consequences and a saving in workload compared with annual assessments.
This paper discusses the lessons learned in application of management procedures and their precursors in this fishery. The high variability in abundance of the two stocks, the trend in their relative abundance, the substantial uncertainties in information, strong pressure to meet socio-economic goals and the conflicting objectives which arose between the directed anchovy and directed sardine fishery are identified as major problems in implementation of procedures and management of the resources. However, the use of management procedures is considered to have led to greatly improved communication with the industry and to substantial input by them into the management process. The procedures and the simulations upon which they were based also enabled consideration of the major sources of uncertainty in understanding of the resource dynamics and facilitated the development of procedures that were robust to them.
It is argued that biological uncertainty greatly exacerbated the problems in application of the procedures but probably cannot be markedly reduced in the near future. Management procedures must be robust to likely variability and uncertainty. Of equal importance are identification and selection of achievable objectives, and allocation to the political decision makers and not to the scientists, of responsibility for determining acceptable trade-offs between conservation and socio-economic goals. Other issues, including the importance of long-term rights and allowance for flexibility in fishing practice, are also highlighted
8.2: Procedures - Biology
As noted by Andrew Kilianski, chief intelligence officer at the joint program executive office for chemical, biological, radiological and nuclear defense, in a July 23 National Defense online article, &ldquoDefense Officials See Increased Threat from Chinese, Russian Chem-Bio Weapons,&rdquo both China and Russia are developing scientific techniques and technologies that &ldquowe haven&rsquot seen before or&hellip that we don&rsquot have a lot of information on.&rdquo
As detailed in the annual Worldwide Threat Assessment of the U.S. Intelligence Community report to the Senate Select Committee on Intelligence, China in particular has increased its economic and resource investments, and deepened political interest in research and innovation to assert growing leverage and power, if not hegemony, in international scientific, biomedical and technological markets.
One of these emerging domains is the growing viability of utilizing synthetic biology to develop novel biological weapons. In 2018, Chinese scientist Dr. He Jiankui used CRISPR/Cas9 germline editing to create human twins with a genetically-induced resistance to HIV. This generated significant ethical and legal controversy, and ultimately led to the World Health Organization determination and assertion that genetic modifications of human germlines are &ldquoirresponsible.&rdquo
However, China has demonstrated that by working at the frontiers of current sciences (i.e. &ndash in some cases, by asserting differing cultural values and ethical norms that guide and govern biomedical research and its uses) they can create, engineer, and foster biological advancements that are as yet unattainable by &mdash and therefore ahead of &mdash other countries.
Avant Garde &mdash and often controversial &mdash synthetic biology synergizes China&rsquos research and of weaponizable biologicals and toxins. Gene editing and other synthetic biology technologies (e.g., artificial proteins) can be employed to increase the potency of a toxin, thereby requiring less to incur a desired effect. This science could be used to genetically modify a low-toxicity bioagent to become more potent and lethal, or could allow the creation of new, unique &mdashand heretofore unknown &mdashagent. Indeed, as James Madsen, lead clinical consultant and clinical laboratory director at the chemical casualty care division at the U.S. Army Medical Research Institute of Chemical Defense, stated in the article: China is the world leader in toxin-based biothreats, and such efforts would only establish that position ever more solidly.
However, the use synthetic biology and gene editing to fortify bioweapons&rsquo development and production is not limited to organic toxins. Prion diseases &mdash or transmissible spongiform encephalopathies (TSEs) &mdash are fatal neurodegenerative disorders that are caused by the misfolding and aggregation of normal prion proteins to then form the disease-causing isoform. Our ongoing research is focused upon prion research, tools, and the ways that increased knowledge and capabilities of genomics and proteomics can enthuse and advance current and near-term future methods and viability of prion synthesis, modification and pathogenicity.
These applications of emerging trends and tools may allow bioweapon programs to produce prion-based agents for kinetic engagements. However, we believe that it is more likely that these agents will be used in non-kinetic engagements to incur multi-domain and multi-scalar disruptive effects that can lead to destructive consequences. Such non-kinetic efforts evoke the types and levels of latent manifestations that are most significant, and therefore of greatest value to strategic competition.
The new methods and tools of synthetic biology can enable the R&D of agents that are not currently listed by the Biological and Toxin Weapons Convention. This makes this R&D &mdash and the agents produced &mdash difficult to surveille, regulate and govern. In light of this, we have called for the update, revision, or new approaches to regnant biochemical weapons&rsquo conventions/treaties and regulatory/governance processes that better reflect and respond to the rapidly changing capabilities fostered by novel techniques and technologies.
Joseph DeFranco is J5 Donovan Group fellow in biowarfare and biosecurity at U.S. Special Operations Command and currently studying biodefense at the Schar School of Policy and Government, George Mason University, Virginia.
James Giordano is professor of Neurology and Biochemistry, chief of the Neuroethics Studies Program, and co-director of the O&rsquoNeill-Pellegrino Program in Brain Science and Global Law and Policy at Georgetown University Medical Center. He currently serves as J5 Donovan Group senior fellow for biowarfare and biosecurity at USSSOM and as an appointed member of the Neuroethics, Legal, and Social Issues Advisory Panel of the Defense Advanced Research Projects Agency.
Candidates must also be able to use the apparatus/instruments to accurately record data (e.g. the use of stopwatch, or thermometer) especially volume, temperature, length and time.
Candidates must also record the results in a table with appropriate headings and units. The results have to be processed and represented as a graph, usually. The identification and comment on key source of errors is common. Drawing appropriate conclusions which coincides with the obtained result.
Candidates must also be able to analyse a practical problem and produce appropriate procedure for the investigation. Included in the planning question should be:
- Approach (A summary of the investigation especially to mention how the dependent variable is measured)
- 3 Fixed dependent variable, 3 Independent variable, 1 dependent variable
- Procedure (a series of steps)
- How to increase reliability