We are searching data for your request:
Upon completion, a link will appear to access the found materials.
I've been using a small egg incubator to grow environmental bacteria in petri dishes. I always wear gloves and glasses when I handle the plates and do Gram staining. How likely is it that bacteria grown on dish could become airborne when I open the incubator or the dishes themselves?
A good practice to minimize the contamination of a Petri dish and of the external environment is by never completely removing the lid of the Petri dish: to access the content of the Petri dish with an inoculation loop, lift the lid on one side, slowly: just high enough for the inoculation loop to get in, get some of the content, and get out.
if you keep the petri dishes covered there will be little or no contamination and there will be negligible e coli aerosol when you open the incubator. as mentioned by @MartinKivana don't open the plates, esp near the incubator. In a sterile environment is better…
if you don't keep the petri dishes covered your experiment will be ruined.
you can wipe it down with bleach once in a while…
so that about covers it.
Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker in bacteria.
Scientists have made many genetic modifications to create bacterial strains that can be more easily transformed and that will help to maintain the plasmid without rearrangement of the plasmid DNA. Additionally, specific treatments have been discovered that increase the transformation efficiency and make bacteria more susceptible to either chemical or electrical based transformation, generating what are commonly referred to as 'competent cells.'
Many companies sell competent cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing. For the highest transformation efficiency, we recommend that you follow the instructions that came with your competent cells.
Last Update: Nov. 13, 2017
Watch the protocol video below to learn how to isolate single bacterial colonies.
- Shaking incubator at 37 °C
- Stationary incubator at 37 °C
- Water bath at 42 °C
- Ice bucket filled with ice
- Microcentrifuge tubes
- Sterile spreading device
- LB agar plate (with appropriate antibiotic)
- LB or SOC media
- Competent cells
- DNA you'd like to transform
Cell Culture Contamination
Biological contamination is the dread of every person working with cell culture. When cultures become infected with microorganisms, or cross-contaminated by foreign cells, these cultures usually must be destroyed. Since the sources of culture contamination are ubiquitous as well as difficult to identify and eliminate, no cell culture laboratory remains unaffected by this concern. With the continuing increase in the use of cell culture for biological research, vaccine production, and production of therapeutic proteins for personalized medicine and emerging regenerative medicine applications, culture contamination remains a highly important issue.
Cell culture is continuing a 60-year trend of increasing use and importance in academic research, therapeutic medicine, and drug discovery, accompanied by an amplified economic impact. 1,2 New therapies, vaccines, and drugs, as well as regenerated and synthetic organs, will increasingly come from cultured mammalian cells. With greater usage and proficiency of cell culture techniques comes a better understanding of the perils and problems associated with cell culture contamination. In the 21st century, there are better testing methods and preventive tools, and an awareness of the risk and effects of contamination requires that cell culturists remain vigilant undetected contamination can have widespread downstream effects.
Biological contamination: a common companion
The chance discovery of penicillin back in 1928 was one of those rare occurrences that most researchers can only dream about. After returning from a summer vacation during which he carelessly left a set of Petri dishes stacked up in a corner of his lab, Alexander Fleming discovered one of the 20th century&rsquos most powerful drugs. Fleming noticed that one of his bacterial cultures was contaminated with a fungus, but the colonies of Staphylococci immediately surrounding the fungus had been destroyed. The fungus was, of course, Penicillium notatum, and Fleming went on to discover antibiotics. This is, however, a very rare example of contamination actually advancing the path of scientific research. For the most part, the contamination of cultures remains every scientist&rsquos worst nightmare. Carolyn Kay Lincoln and Michael Gabridge summed up the problem in 1998: &ldquoCell culture contamination continues to be a major problem at the basic research bench as well as for bioproduct manufacturers. Contamination is what truly endangers the use of cell cultures as reliable reagents and tools.&rdquo 3
The biological contamination of mammalian cell cultures is more common than you might think. Statistics reported in the mid-1990s show that between 11 percent and 15 percent of cultures in U.S laboratories were infected with Mycoplasma species. 4 Even with better recognition of the problem and more stringent testing of commercially prepared reagents and media, the incidence of mycoplasma growth in research laboratory cultures was 23 percent in one recent study, 5 and in 2010 an astonishing 8.45 percent of cultures commercially tested from biopharmaceutical sources were contaminated with fungi and bacteria, including mycoplasma. 6
In the research laboratory, contamination is not just an occasional irritation, but it can cost valuable resources including time and money. Ultimately, contamination can affect the credibility of a research group or particular scientist publications sometimes must be withdrawn due to fears about retrospective sample contamination or reported results that turn out to be artifacts. In biopharmaceutical manufacturing, contamination can have an even more dramatic effect when entire production runs must be discarded. It is extremely important, therefore, to understand how sample contamination can occur and what methods are available to limit and, ultimately, prevent it.
What causes biological contamination?
Biological contaminants can be divided into two subgroups depending on the ease of detecting them in cultures, with the easiest being most bacteria and fungi. Those that are more difficult to detect, and thus present potentially more serious problems, include Mycoplasmas, viruses, and cross-contamination by other mammalian cells.
Bacteria and fungi
Bacteria and fungi, including molds and yeasts, are ubiquitous in the environment and are able to quickly colonize and flourish in the rich cell culture milieu. Their small size and fast growth rates make these microbes the most commonly encountered cell culture contaminants. In the absence of antibiotics, bacteria can usually be detected in a culture within a few days of contamination, either by microscopic observation or by their direct effects on the culture (pH shifts, turbidity, and cell death). Yeasts generally cause the growth medium to become very cloudy or turbid, whereas molds will produce branched mycelium, which eventually appear as furry clumps floating in the medium.
Mycoplasmas are certainly the most serious and widespread of all the biological contaminants, due to their low detection rates and their effect on mammalian cells. Although mycoplasmas are technically bacteria, they possess certain characteristics that make them unique. They are much smaller than most bacteria (0.15 to 0.3 &mum), so they can grow to very high densities without any visible signs. They also lack a cell wall, and that, combined with their small size, means that they can sometimes slip through the pores of filter membranes used in sterilization. Since the most common antibiotics target bacterial cell walls, mycoplasmas are resistant.
Mycoplasmas are extremely detrimental to any cell culture: they affect the host cells&rsquo metabolism and morphology, cause chromosomal aberrations and damage, and can provoke cytopathic responses, rendering any data from contaminated cultures unreliable. In Europe, mycoplasma contamination levels have been found to be extremely high&mdashbetween 25 percent and 40 percent&mdashand reported rates in Japan have been as high as 80 percent.4 The discrepancy between the U.S. and the rest of the world is likely due to the use of testing programs. Statistics show that laboratories that routinely test for mycoplasma contamination have much lower incidence once detected, contamination can be contained and eliminated. Testing for mycoplasma should be performed at least once per month, and there is a wide range of commercially available kits. The only way to ensure detection of species is to use at least two different testing methods, such as DAPI staining and PCR. 5
Like mycoplasmas, viruses do not provide visual cues to their presence they do not change the pH of the culture medium or result in turbidity. Since viruses use their host for replication, drugs used to block viruses can also be highly toxic for the cells being cultured. Viruses that cause damage to the host cell do, however, tend to be self-limiting, so the major concern for viral contamination is their potential for infecting laboratory personnel. Those working with human or other primate cells must use extra safety precautions.
Other mammalian cell types
Cross-contamination of a cell culture with other cell types is a serious problem that has only recently been considered alarming. 7,8 An estimated 15 percent to 20 percent of cell lines currently in use are misidentified 9,10 , a problem that began with the first human cell line, HeLa, an unusually aggressive cervical adenocarcinoma isolated from Henrietta Lacks in 1952. HeLa cells are so aggressive that, once accidentally introduced into a culture, they quickly overgrow the original cells. But the problem is not limited to HeLa there are many examples of cell lines that are characterized as endothelial cells or prostate cancer cells but are actually bladder cancer cells, and characterized as breast cancer cells but are in fact ovarian cancer cells. In these cases, the problem occurs when the foreign cell type is better adapted to the culture conditions, and thus replaces the original cells in the culture. Such contamination clearly poses a problem for the quality of research produced, and the use of cultures containing the wrong cell types can lead to retraction of published results.
Sources of biological contaminants in the lab
In order to reduce the frequency of biological contamination, it is important to understand how biological contaminants can enter culture dishes. In most laboratories, the greatest sources of microbes are those that accompany laboratory personnel. These are circulated as airborne particles and aerosols during normal lab work. Talking, sneezing, and coughing can generate significant amounts of aerosols. Clothing can also harbor and transport a range of microorganisms from outside the lab, so it is crucial to wear lab coats when working in the cell culture lab. Even simply moving around the lab can create air movement, so the room must be cleaned often to reduce dust particles.
Certain laboratory equipment, such as pipetting devices, vortexers, or centrifuges without biocontainment vessels, can generate large amounts of microbial-laden particulates and aerosols. Frequently used laboratory equipment, including water baths, refrigerators, microscopes, and cold storage rooms, are also reservoirs for microbes and fungi. Improperly cleaned and maintained incubators can serve as an acceptable home for fungi and bacteria. Overcrowding of materials in the autoclave during sterilization can also result in incomplete elimination of microbes.
Culture media, bovine sera, reagents, and plasticware can also be major sources of biological contaminants. While commercial testing methods are much improved over those of earlier decades, it is paramount to use materials that are certified for cell culture use. Cross-contamination can occur when working with multiple cell lines at the same time. Each cell type should have its own solutions and supplies and should be manipulated separately from other cells. Unintentional use of nonsterile supplies, media, or solutions during routine cell culture procedures is the major source of microbial spread.
Contamination is a prevalent issue in the culturing of cells, and it is essential that any risks are managed effectively so that experiment integrity is maintained. Antibiotics can be used for a few weeks to ensure resolution of a known microbial contamination however, routine use should be avoided. Regular inclusion of antibiotics not only selects for resistant organisms, but also masks any low-level infection and habitual mistakes in aseptic technique.
The best approach to fighting contamination is for each person to keep records of all cell culture work including each passage, general cell appearance, and manipulations including feeding, splitting, and counting of cells. If contamination does occur, make a note of the characteristics and the time and date. In this way, any contamination can be pinpointed at the time it occurs and improvements can be made to aseptic techniques or lab protocols. In the next article of this series, we explore in more detail effective measures for contamination prevention, in particular the key role of the CO2 incubator.
Another reason for incubating at different temperatures is promoting the growth of a target group of bacteria. For example, although both pathogens and environmental bacteria found indoors are mesophiles, pathogens will grow faster than environmental strains at 37 degrees Celsius (98 degrees Fahrenheit), the temperature of the human body. The opposite is true at 25 C (77 F), the temperature of most buildings. Since student teaching laboratories often wish to prevent pathogen growth, cultures are often incubated at 25 C (77 F).
Successful incubation is an essential step in your everyday workflows. Thermo Scientific Microbiological and Refrigerated Incubators are designed with your samples in mind to produce results you can count on.
To browse our complete portfolio or let one of our experts help you make a selection.
Heratherm Microbiological Incubators
Heratherm Refrigerated Incubators
Precision Low Temperature Refrigerated Incubator*
Incubators for microbiological studies are available with different technologies, to address the specific incubation temperatures that are needed for the application.
Microbiological incubators, also called "heat-only" or "standard" incubators have heating elements, and can provide incubation temperatures that are above ambient temperature only. If the laboratory has an ambient of about 22°C, they can only address incubation temperatures above about 27°C or even 30°C.
Refrigerated incubators, also called "cooling" incubators have cooling and heating, and can provide a wider temperature range - offering also temperatures close to ambient or even below ambient. They usually cover also the incubation temperature range above ambient - as the "microbiological" or "heat-only" incubators do. Due to the more complex technology used, a refrigerated incubator is a higher investment.
What are the guidelines for culturing brine shrimp?
The benefits of feeding live artemia are well known and accepted in the aquarium community. Alternatively, there are many convenient and well-formulated artificial, inert diets that purport to completely eliminate the need for such live food. These prepared diets and, more importantly, the specific amino acids, lipids, and vitamins they contain are, if not complete replacements for live feed, often necessary additions to a single species diet lacking in one or more of the essential nutrients.
That having been said, seldom does a soggy, inanimate particle of gelatinized starch and dried fishmeal ignite the feeding response in fish like the herky-jerky swimming antics of a live brine shrimp. For this reason, live brine shrimp will always be an integral part of the solution for sustaining healthy aquarium populations.
In addition to moving about the water column, live brine shrimp have a number of other useful traits, namely:
- They are soft and easily digestible and contain enzymes that help fish to better utilize other feeds
- They are high in protein, ranging from 55% to 60% protein by dry weight, supporting rapid weight gain in young fish
- They can be enriched with other feeds or additives, a process often referred to as "bio-encapsulation" in order to deliver HUFAs, antibiotics, or other nutrients to the target species (see SELCO)
- They can be fed to both marine and freshwater fish, surviving and swimming for hours &mdash even in fresh water
- They originate in hyper-saline biotopes and, therefore, they are seldom vectors for diseases that affect fish
- They grow quickly, multiplying in weight 500-fold in three to four weeks and increasing in size from 450 microns to 1.5 centimeters in length.
Yet, raising brine shrimp to maturity in useful numbers is not an easy task and you can expect to spend as much time at it, if not more, as you would breeding and caring for baby fish &mdash often with less-than-hoped for results. The following primer is designed to help obviate the need to commit the most frequent mistakes &mdash most often, the mistakes of overstocking, overfeeding, underfeeding, inadequate aeration, under-filtration, and providing inappropriate feeds.
Given the myriad ways to inadvertently kill these critters, even in a comfortable, controlled environment, it seems counter-intuitive, if not downright discouraging to the aquarist, that an animal of prehistoric pedigree can, in its natural setting, be left high and dry in the summer, desiccated for months under the hot sun, forcibly removed to a faraway clime in the gut of an avian migrant, re-deposited in a hyper-saline lake devoid of life, and subjected to subfreezing temperatures, only to emerge from its capsule to thrive again and even propagate.
Without further rumination, let's assume that we've successfully hatched the eggs and wish to culture the brine shrimp. Can we not, as our well-intentioned Webmaster asked, just send them off to a French finishing school?
The culture tank can be as simple as a 5-gallon bucket or as involved as a $500 Kreisel tank. The important design considerations, when choosing or building a culture tank, are to allow for temperature control adequate aeration (to maintain dissolved oxygen levels as well as to suspend food particles) internal or external water filtration and/or partial water replacement and the concentration and evacuation of detritus, mortalities, and fecal matter (through screened drainpipes or siphoning). Culture systems are varied, from batch or static systems, to sophisticated flow-through tanks for high-density culture. If the intent is simply to observe a small number of brine shrimp, an aquarium with a sub-sand/mud filter and one or two directional airlift standpipes is ideal.
To improve your chances of success, start with stocking densities of 1,000 animals per liter or less.
How does one count 1,000 miniscule baby brine shrimp? The easiest way to manage artemia counts is to sample from a randomly distributed population by extracting small aliquots of the whole, counting, and then extrapolating. Let's assume that we began with one gram (about 1/2 teaspoon) of an 80% hatch-out quality egg. If, after 24 hours' incubation, we recover most of the newly hatched baby brine shrimp, we would have upwards of 200,000 baby brine shrimp!
To test our theory of aliquot sampling, transfer all the animals into a one-liter bottle containing clean seawater with aeration. The aeration will keep the brine shrimp in suspension, and thus randomly distributed throughout the cone or bottle. Using a one-milliliter pipette, extract one milliliter of water, and with it, approximately 1/1000th of the total population (1 liter = 1,000 milliliters). If our sampling technique is adequate (replicates are advisable), we should have about 200 animals in our aliquot sample. If a pipette is not available, a calibrated eyedropper can be used to pull a measured sample from the bottle.
Note: If this process has already exceeded your tolerance for tedium, you may want to consider stopping here, enriching all the newly harvested brine shrimp contained in the one-liter bottle with SELCO, and feeding the fortified baby brine shrimp to your fish or seahorses. But, if you insist on bigger and beefier brine shrimp, then read on. Just remember &mdash you had your chance!
The preferred salinity range for culturing brine shrimp is 35-40 ppt (specific gravity 1.024-1.028). Unlike in the preparation of hatching solutions, where household brands of baking salt, kosher salt, and solar salt are adequate, culture water should be pre-mixed using an aquarium-grade marine salt. Remember to pre-mix and stock additional water for use later. You'll need it!
The initial pH should be between 7.5 and 8. The pH is likely to fall during the culture period and can be adjusted upward with the addition of baking soda or NaHCO3. Monitor pH regularly and adjust as needed.
Artemia are generally tolerant of low dissolved oxygen levels. Oxygen stress is often indicated by a reddening of the animals caused by the increased presence of hæmoglobin. Providing adequate aeration to keep food in suspension usually eliminates any DO (dissolved oxygen) concerns. Keep in mind that small bubbles are more efficient vehicles for oxygen transfer, but that very fine bubbles actually foul the swimming appendages and interfere with feeding. If the DO level falls below 2.5 mg/l-1, add additional airstones.1
Temperature should be maintained at between 20° Celsius and 25° Celsius (68°F-79°F). Remember that replacement water should be of similar temperature to avoid thermal shock.
Artemia are continuous, non-selective filter feeders.2 The most difficult challenge in culturing artemia is providing appropriately sized feed in sufficient concentrations without unduly compromising water quality. Fortunately, there are a number of easily obtained feeds that are optimal in terms of both size (less than 20 microns) and nutritional content.
Tank design and aeration play an important role in the distribution of feed throughout the water column. Feed must be kept in suspension in order to be utilized. This is accomplished by the use of directional airlifts, air stones, and return water flows. When using dry feeds, better food suspension is achieved by premixing the feed with clean seawater.
Nitrogen levels should be monitored. NO2-N levels should be kept below 320 mg/l-1.3 Water quality is controlled by a combination of mechanical filtration, either external or internal, and or dilution with new, clean water.
In order to maintain adequate water quality, the suspended solids, uneaten food, fecal matter, and detritus must be removed regularly. This presents the conundrum: How does one efficiently remove these pollutants without also removing the food? Unfortunately, there is not an easy answer. The approaches described in literature are more art than science, or glossed over altogether. Certain losses in food density due to filtration are unavoidable. Compromises often entail filtering out large flocks only (allowing food and suspended solids to pass through), allowing flocks to settle or concentrate near effluent drains before removing, using longer water retention times, and/or alternating between intermittent filtering and feeding cycles.
As animals grow, filters with larger mesh sizes can be used. A typical filter at the outset will have openings of 100 microns. These openings can be increased to 350 microns when the animals are about two weeks old. Filters must be cleaned regularly. Placing air stones in front of effluent filters will help prevent blinding of the filter. Obviously, increasing rates of water exchange and increasing filter opening sizes will necessitate a commensurate increase in feeding rates in order to maintain desired food cell densities.
Culture density, food cell density, and animal health can be checked by routinely removing and examining a beaker of water and holding it against a light for close inspection. It is possible to note the fullness of the gut and determine if the animals are adequately fed. Food cell density can also be measured by inserting a Secchi disc into the tank water to measure clarity. The depth that the Secchi disc is lowered into the water before it is obscured is observed and recorded. Feeding rates and exchange rates are maintained at a level that works for your particular system.
The preferred feed for artemia is cultured, live diatoms. A number of species have been used successfully, including Nannochloropsis sp., Tetraselmis sp., and Dunaliella sp. Providing live diatoms, of course, entails a duplicate effort commensurate with the number of artemia to be fed. As stated before, brine shrimp are continuous feeders and, at high densities, quickly clear water of diatoms. Reliance on live diatom cultures, though practicable, should be done so with substitute frozen or dry feeds within easy reach should your algae cultures crash.
One of the best choices in readily available feeds that we have found for culturing artemia are the cryo-preserved algae pastes, particularly Nannochloropsis sp. or the proprietary mix, Tahitian Blend, which contains several alga species and stabilized vitamin C. These pastes are non-viable, highly concentrated algal cells that can be administered to the culture tank drop-wise. Using cryo-pastes of known cell density can allow the culturist to quickly harmonize feeding levels with the density and growth of the artemia population.
Other feeds that have been used successfully to culture artemia are the spray-dried, single-celled yeasts, most notably Torula. Other feeds that have been used to culture brine shrimp are micronized forms of rice bran, corn bran, and soybean.4 These feeds are often used in combination with other approaches. The proper sizing of particles can be attained by micronizing (using an electric blender) brans with seawater and filtering through a 250-mesh or finer bag. Spray-dried Arthrospira platensis (formerly Spirulina platensis) has also been used to sustain brine shrimp. Feeds that readily leach nutrients into the water should be avoided, as they will contribute to high bacterial loads, increased oxygen demand, and fouling of swimming appendages.
As stated before, there are many and varied systems that have been devised for the on-growing of brine shrimp. For high-density culture (10,000+ animals per liter), survival requires robust mechanical filtration and water exchange &mdash in effect, a raceway system with ancillary treatment and filtration equipment. Lower density (1,000 animals/lt.) "batch" systems rely on a combination of regular water exchange or dilution with clean seawater and regular removal of detritus. In the batch system, feeding rates are lowered to compensate for longer water retention times, oftentimes resulting in slower growth. In either system, water quality is enhanced by the addition of a protein skimmer.
It is not uncommon for filamentous Leucothrix bacteria to emerge in the protein-rich culture environment. Vibrio sp. bacteria and other infectious diseases may present. These outbreaks may be treated with antibiotics and/or controlled by increasing salinity. It is important to use disinfected cysts and to routinely disinfect culture apparatus with a hypochlorite solution.
As suggested earlier, producing live adult artemia in sufficient numbers to feed numerous fish tanks or seahorse pens requires considerable work. The demands of feeding artemia and cleaning filters are non-relenting 24/7. On the other hand, low-density culture of artemia is rewarding and less trying. Our best advice is to start small and scale up gradually.
1Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper 361, Lavens, P and Sorgeloos, P. 1996, p. 168.
School microbiology will generally be safe, but before any practical activity is undertaken, risks must be assessed.
Each individual (students, technicians or teachers) embarking on a practical activity is responsible for his or her health and safety and that of others affected by the work.
Risk assessment will involve comparing the steps involved in an intended activity with procedures suggested in model risk assessments. This will identify the safety precautions that need to be taken in the context of the level of work and, possibly, the need to amend the procedure so that the risks to health and safety from any hazard/s are minimised.
Local rules must also be complied with. Of greatest importance in risk assessment is a consideration of the skills and behaviour of the students about to tackle a practical activity a procedure that is safe for one group of individuals may need to be modified with a different class.
Also important is ensuring that a procedure is safe for pupils, but also does not endanger the health and safety of technicians or teachers during preparation or disposal. In deciding on the appropriate precautions to adopt, it is prudent that all cultures are treated as potentially pathogenic (for example, because of possible contamination).
Emergency procedures, such as dealing with spills, should also be considered.
Cell-Based Flu Vaccines
Cell-based flu vaccine production does not require chicken eggs because the vaccine viruses used to make vaccine are grown in animal cells.
What are cell-based flu vaccines?
&lsquoCell-based&rsquo refers to how the influenza (flu) vaccine is made. Most inactivated flu vaccines are produced by growing flu viruses in eggs. The flu viruses used in the cell-based vaccines are grown in cultured cells of mammalian origin instead of in hens&rsquo eggs.
Flucelvax Quadrivalent is the only cell-based inactivated flu vaccine that has been licensed by the FDA for use in the United States.
Who can get Flucelvax Quadrivalent?
Flucelvax Quadrivalent is licensed for use in people 4 years and older.
Why has a cell-based flu vaccine been developed?
Cell-based flu vaccine production does not use flu viruses grown in eggs and, therefore, is not dependent on the supply of eggs. In addition, cell-based flu vaccines that use cell-based candidate vaccine viruses (CVVs) have the potential to offer better protection than traditional, egg-based flu vaccines. The viruses used to make cell-based vaccines may be more similar to circulating &ldquowild&rdquo flu viruses than the viruses used to make egg-based vaccines. In one study published in the Journal of Infectious Diseases external icon among Medicare beneficiaries 65 years and older, cell-based vaccine provided greater protection against flu-related hospitalizations than standard-dose, egg based vaccine.
For the 2020-2021 flu season, all four flu viruses used in the Flucelvax Quadrivalent are cell-derived, making the vaccine egg-free.
How is the cell-based vaccine manufacturing process different than the traditional egg-based manufacturing process?
The cell-based vaccine manufacturing process uses animal cells (Madin-Darby Canine Kidney, or MDCK cells) as a host for the growing flu viruses instead of fertilized chicken eggs. For the 2020-2021 season, the viruses provided to the manufacturer to be grown in cell culture are cell-derived rather than egg-derived. Learn more about the cell-based flu vaccine manufacturing process on CDC&rsquos How Flu Vaccines are Made web page.
What is the significance of FDA approving cell-based candidate vaccine viruses for use in the Flucelvax Quadrivalent cell-based flu vaccines?
Growing flu viruses in eggs can introduce changes (called egg-adapted changes) that can cause differences between the viruses in the vaccine and the ones that are circulating. These changes may have important implications for the body&rsquos immune response to vaccination. For example, egg-adapted changes could cause the body&rsquos immune system to produce antibodies that are less effective at preventing disease caused by the specific flu viruses in circulation. FDA&rsquos approval of cell-based CVVs for use in cell-based flu vaccines could possibly improve the effectiveness of cell-based flu vaccines.
What are the possible benefits of using cell-based flu vaccines?
Observational studies have shown greater protection against flu or flu-like illness among people who received Flucelvax compared to those who received standard-dose egg-based vaccines.
A potential advantage of cell culture technology is that it might permit faster start-up of the vaccine manufacturing process in the event of a pandemic. The cells used to manufacture Flucelvax Quadrivalent are kept frozen and &ldquobanked.&rdquo Cell banking ensures an adequate supply of cells is readily available for vaccine production. Growing the flu viruses in cell culture for the manufacture of Flucelvax Quadrivalent is not dependent on an egg supply. Cell-based flu vaccines that are produced using CVVs have the potential to be more effective than traditional egg-based flu vaccines.
What were the results of the clinical trials using cell-based technology?
A clinical trial of the previous trivalent formulation of Flucelvax demonstrated effectiveness and safety among persons 18 through 49 years old. In immunogenicity studies among people 18 years and older and 4 through 17 years old, Flucelvax Quadrivalent was found to produce a similar immune response to the trivalent formulation. Post-vaccination symptoms were typical of those seen with other injectable flu vaccines.
Has cell-based technology been used before?
Cell culture technology has been used to produce other U.S.-licensed vaccines, including vaccines for rotavirus, polio, smallpox, hepatitis, rubella and chickenpox.
Cell-based flu vaccines have been approved for use in many European countries.
Why use Drosophila?
Teachers should use fruit flies for high school genetic studies for several reasons:
1. They are small and easily handled.
2. They can be easily anesthetized and manipulated individually with unsophisticated equipment.
3. They are sexually dimorphic (males and females are different), making it is quite easy to differentiate the sexes.
4. Virgins fruit flies are physically distinctive from mature adults, making it easy to obtain virgin males and females for genetic crosses.
5. Flies have a short generation time (10-12 days) and do well at room temperature.
6. The care and culture of fruit flies requires little equipment, is low in cost and uses little space even for large cultures.
By using Drosophila, students will:
1. Understand Mendelian genetics and inheritance of traits
2. Draw conclusions of heredity patterns from data obtained
3. Construct traps to catch wild populations of D. melanogaster
4. Gain an understanding of the life cycle of D. melanogaster, an insect which exhibits complete metamorphosis
5. Construct crosses of caught and known wild- type and mutated flies
6. Learn techniques to manipulate flies, sex them, and keep concise journal notes
7. Learn culturing techniques to keep the flies healthy
8. Realize many science experiments cannot be conducted and concluded within one or two lab sessions
National standards covered in these lessons:
1. Organisms require a set of instructions for specifying traits (heredity)
2. Hereditary information is located in genes.
3. Combinations of traits can describe the characteristics of an organism.
1. Identify questions and concepts that guide scientific investigations
2. Design and conduct scientific investigations
3. Formulate and revise scientific explanations and models using logic and evidence
4. Communicate and defend a scientific argument
The genetics of Drosophila are well documented and several public-domain web sites feature the complete annotated genome. Therefore, those teachers or students wishing to see where their mutations occur have a ready reference available.
Since Drosophila has been so widely used in genetics, there are many different types of mutations available for purchase. In addition, the attentive student may find mutations within their own wild-caught cultures since, due to a short generation time, mutations are relatively common compared to other animal species.
Genus: Drosophila (“dew lover”)
Species: melanogaster (“dark gut”)
Life cycle of Drosophila melanogaster
Drosophila melanogaster exhibits complete metamorphism, meaning the life cycle includes an egg, larval (worm-like) form, pupa and finally emergence (eclosure) as a flying adult. This is the same as the well-known metamorphosis of butterflies. The larval stage has three instars, or molts.
Day 0: Female lays eggs
Day 1: Eggs hatch
Day 2: First instar (one day in length)
Day 3: Second instar (one day in length)
Day 5: Third and final instar (two days in length)
Day 7: Larvae begin roaming stage. Pupariation (pupal formation) occurs 120 hours after egg laying
Day 11-12: Eclosion (adults emerge from the pupa case).
• The generation time of Drosophila melanogaster varies with temperature. The above cycle is for a temperature of about 22°C (72°F). Flies raised at lower temperature (to 18°C, or 64°F) will take about twice as long to develop.
• Females can lay up to 100 eggs/day.
• Virgin females are able to lay eggs however they will be sterile and few in number.
After the eggs hatch, small larvae should be visible in the growing medium. If your media is white, look for the small black area (the mouth hooks) at the head of the larvae. Some dried premixed media is blue to help identify larvae however this is not a necessity and with a little patience and practice, larvae are easily seen. In addition, as the larvae feed they disrupt the smooth surface of the media and so by looking only at the surface one can tell if larvae are present. However, it is always a good idea to double check using a stereo microscope. After the third instar, larvae will begin to migrate up the culture vial in order to pupate.
Practical Work for Learning
Incubating the plates to promote growth of microbes is an essential part of any microbiology investigation. Incubating in aerobic conditions, and below human body temperature, reduce the risk of encouraging microorganisms (particularly bacteria) that could be pathogenic to humans. Taping the lids on reduces the chance that students will open plates when viewing, but there are details below of how to kill plates completely if this is still a significant risk.
Health & Safety and Technical notes
Carry out a full risk assessment before starting any microbiology work (see note 1 for more details).
2 Keep plates at room temperature or incubate at 20-25 °C for 2-3 days. Fungi grow more successfully at lower temperatures. Do not incubate at human body temperature (or above 30 °C) – this reduces the risk of culturing microbes that are pathogens to humans.
3 Reducing the temperature to 4 °C will slow the growth of any cultures – so you can show your students a 2-3 day growth if your lessons are a week apart.
4 All inoculated plates must be taped before incubation to ensure they cannot be opened accidentally. Do this by fixing with 2 or 4 short strips of adhesive tape at opposite edges of the dish. Do not seal completely as this may promote the growth of anaerobic pathogens or prevent normal growth by restricting diffusion of oxygen. See CLEAPSS Laboratory Handbook 15.2.10.
5 Plates are incubated upside down (agar up), so that condensation does not drip onto the plate and interfere with the developing microbes.
6 You might replace lids if condensation makes viewing difficult, so label plates on the bottom – with Chinagraph or wax pencils, permanent marker pens or a small adhesive label at one edge.
7 Count the plates out and in again to ensure that you have collected all the plates at the end of a lesson.
8 You can seal plates around the whole circumference just before viewing if you think there is any risk that your students will open the plates. Or you can stop the growth of a culture completely by placing a piece of filter paper into the lid of the inverted plate. Add a little 40% methanal solution carefully to soak the filter paper and replace the base. Leave for 24 hours. Remove the filter paper, remove any surplus liquid, and reseal the plate. See CLEAPSS Laboratory Handbook section 15.2.11. On Hazcard 063, methanal is described as toxic at this concentration and a category 3 carcinogen.
Society for General Microbiology – source of Basic Practical Microbiology, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms.
MiSAC (Microbiology in Schools Advisory Committee) is supported by the Society for General Microbiology (see above) and their websites include more safety information and a link to ask for advice by email.
(Websites accessed October 2011)
© 2019, Royal Society of Biology, 1 Naoroji Street, London WC1X 0GB Registered Charity No. 277981, Incorporated by Royal Charter