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Frequently asked questions
Chemistry, Basic definitions, PH, Molarity…
For good experimental practice in the laboratory, it is imperative to consider the temperature of your reagents. Always allow your reagents to return to room temperature in the laboratory (generally between 18 and 22°C) before starting your experimental protocol. This will ensure better reproducibility of your results, considering not only concentrations but also temperature and reaction time parameters.
Water is essential to life science research, but for many scientists, the most important reflection on this most widely used laboratory reagent is “how much do I need?” Because water is capable of acting as a solvent for many substances and reagents (aqueous solution), beware it can become contaminated. Water can also be a dilution medium.
Even within the same laboratory, the water used in one experiment may differ from that used in another. There are a multitude of methods, devices and means of producing purified water, so we’ll need to specify the means of producing this water. Not all water is the same, so consider water as a laboratory reagent and examine it accordingly. Understanding the importance of water quality and monitoring it closely is important to ensure experimental reproducibility over several days, months and even years.
The CAS number (or CAS registry number) of a chemical, polymer, biological sequence or alloy is its unique registration number in the database of the Chemical Abstracts Service (CAS), a division of the American Chemical Society (ACS). CAS assigns these numbers to each chemical substance described in the documentation. CAS also maintains and markets a database of these substances, the CAS Registry.
The American Chemical Society (ACS), with over 150,000 members, is one of the world’s largest scientific organizations and one of the world’s leading sources of authoritative scientific information on chemistry.
It publishes references, orders and describes every ACS product according to a standardized method. If you need more detailed information about a chemical product with an ACS label, you can find its exact composition in the database available free of charge on the https://www.acs.org/content/acs/en.html website.
Microfiltered solutions are produced by vacuum filtration on a membrane (filtration device). There are many different types of membrane (diameter, porosity, materials, etc.)
Membranes used to filter an aqueous solution are made from a cellulose-based filter (cellulose ester), with a diameter adapted to your filtration device and a porosity of 0.4µ or 0.2µ (the 0.2µ porosity also enables bacteria to be retained (hence the sterilization method).
Cellular compartments operate at pH levels close to 7. There is no universal pH for fixation.
A stock solution is generally a concentrated solution that can be diluted to obtain less concentrated daughter solutions. Example: prepare a 0.2M stock solution to obtain a 0.1M phosphate buffer (daughter solution), a 0.4 M stock solution to obtain a 0.2M daughter solution.
All products on the market generally have an expiration date. This doesn’t necessarily mean you can’t use them after that date. The expiration date is the date on which the supplier’s warranty on your product expires.
We’ve all tested this, sometimes using chemicals that have been out of date for several years, and the result was excellent (it generated less waste). It’s important to remember that product shelf-life is highly dependent on packaging and storage.
EM-grade aims to be an eco-responsible player in the electron microscopy sector, promoting the rational use of chemicals which have a dual impact on our environment and our health.
If we can’t do without certain sometimes toxic products, then let’s consume better and less often.
That’s why we prefer to sell products in small volumes and small packaging sizes, to avoid stocking up for years on end in laboratory waste.
Laboratory professionals are not big consumers of small packs for two main reasons:
Fear of running out of product during handling operations
The higher cost of products, as opposed to bulk purchases.
EM grade guarantees shipment within 24 hours (no breakdowns) and reasonable prices for all quantities.
The Periodic Table of the Elements, also known as the Mendeleyev Table, the Periodic Table or simply the Periodic Table, represents all the chemical elements, ordered by ascending atomic number and organized according to their electronic configuration, which underlies their chemical properties.
The conception of this table is generally attributed to the Russian chemist Dimitri Ivanovitch Mendeleev.
A neutral (inert) gas is a stable gas (meaning it already has 8 valence electrons in its outer shell and doesn’t need to react to reach these 8 electrons). These gases are also known as noble or rare gases. They do not initiate chemical reactions on their own (nitrogen is an inert gas, as is Argon, the most widely used in EM-grade conditions).
The laboratory water
When the buffer has a high concentration, from 0.4 molar upwards, it can crystallize to some extent when stored in the refrigerator. This condition is reversible, as the crystals will dissolve within a few hours at room temperature. You can reheat it to accelerate the disappearance of the crystals. This crystallization does not change the chemical properties of the buffer.
DPBS (Dulbecco’s Phosphate Buffered Saline)
PBS (Phosphate Buffer Saline). Due to osmolarity constraints associated with the fixative, or for rinsing purposes, it is generally prepared at 0.1M or 0.2M at a pH of around 7 to 7.4, to compensate for the slight acidity released by cells during fixation.
DPBS (developed by Dulbecco) may contain CaCl2 and MgCl2 additives to facilitate cell detachment in culture. If DPBS does not contain these additives, it can be replaced by PBS.
Calcium and magnesium facilitate cell binding and agglutination (facilitating cell recovery in dish culture).
Please note :
- D-PBS is generally slightly lower in phosphate concentration than PBS,
- DPBS may or may not include calcium and/or magnesium. Experimental needs will dictate which solution to use.
For example, if in a particular experiment the enzymatic activity of trypsin is to be measured, the calcium and magnesium sometimes included in DPBS may distort the results, so plain PBS would be preferable.
Phosphate buffers are the most widely used solutions in biology, as they are non-toxic.
However, this non-toxicity may encourage the development of micro-organisms in the medium term. This disadvantage can be overcome by preparing stock solutions with high concentrations of 0.4M, 1M and higher, as micro-organisms do not proliferate in high salt concentrations (with a few exceptions).
Phosphate-based buffers become contaminated after a few months (2 to 3 months max.), as they are vital. They can be decontaminated and continue to be used simply by filtering at 0.22µ (syringe filter, etc.). Store at 4 – 8°C.
It is not a vital buffer, as it contains traces of toxic elements such as arsenic.
However, it is still widely used when fixing cells (since fixing kills the cell). In this case, cacodylate’s toxicity is no longer a drawback. On the contrary, its toxicity becomes an advantage, thanks to its ease of preparation. It has a long shelf-life (toxic to micro-organisms) and does not require sterilization.
The usefulness of imidazole-buffered osmium tetroxide as a lipid preservative in transmission electron microscopy is highly recommended, as imidazole allows lipid retention and maintenance even after alcoholic dehydration. As an example, Rat liver and other tissues were perfusion-fixed with glutaraldehyde and post-fixed with osmium-imidazole, and the appearance of lipid droplets was compared to that after fixation in aqueous osmium tetroxide unbuffered with imidazole. Prominent electron-opaque staining of lipid droplets and lipoprotein particles was noted after post-fixation with 1% 0.1M osmium-imidazole, pH 7.5, for 30 min. (more details see protocol).
Better manage your chemical inventories with a simple quantified inventory to anticipate your purchases.
Group your purchases together to save money, but above all to limit the carbon impact on the planet.
We often negotiate delivery costs without considering the impact on our environment.
EM-grade will give you a discount on delivery costs for all grouped orders.
In 1966, Norman Good and his colleagues set out to define the best buffers for biochemical systems.
In 1980, Good and his colleagues identified twenty buffers that set the standard for use in the biological and biochemical research we now use in microscopy to fix tissues.
Sometimes the storage of your vital tampons (phosphate, PHEM,…), their expiration date (guarantee) exceeded, can become contaminated by the proliferation of bacteria or mold. This state of contamination in no way diminishes the properties of your tampons. Simply refilter to 0.22µm or autoclave your buffer solution.
Once treated, your swabs are safe to use.
EM-grade means that this product is qualified for use in the preparation of samples to be examined under an electron microscope.
An EM. grade product must be:
- high chemical purity and distilled
- sealed in a neutral atmosphere (saturated with a neutral gas such as Argon or Nitrogen) without contact with air, either oxygen or CO2 (AirLess)
For small volumes, seal in an ampoule or Airless bottle.
For larger volumes, we inject argon or nitrogen, saturating the liquid by bubbling the bottled solution to replace the oxygen or carbon dioxide contained in the water.
But when you open the bottle, you lose the neutral atmosphere. That’s why we sell small volumes to avoid waste.
There’s no risk of running out of your products for your experiments, as they’re produced on the day you order them and dispatched immediately.
Air contains Oxygen, the main oxidizing agent in nature, which degrades your product’s components.
Oxygen and a little CO2 (Air) are replaced by neutral gases such as Argon or Nitrogen.
This allows your product to keep longer, if you don’t open the bottle. For this reason, it’s best to buy small volumes, so that you always have effective products when you need them.
Fixing cells means preserving them in a state as close as possible to their living state. Chemicals are used to fix proteins and other cell components.
The fixative solution is generally a mixture of fixative, buffer and various additives when specified in your protocol.
Ready-to-use: i.e. the concentration of fixatives, molarity and type of buffer adjusted to the required pH (according to your protocol), taking into account the final osmolarity of the solution, from a few hundred milliOsmoles for plants, invertebrates and mammals, to a thousand milliOsmoles for aquatic or marine species.
Aldehydes are the most commonly used fixatives. They are used to stabilize the finest structural details of cells and tissues prior to examination by light or electron microscopy.
They can be referred to as primary fixatives, such as ParaFormAldehyde (PFA), Glutaraldehyde or their Karnovsky mixture.
Mixtures containing Paraformaldehyde (PFA) and glutaraldehyde
The combination of Paraformaldehyde with glutaraldehyde as a fixative for electron microscopy benefits from the rapid penetration of small HCHO molecules, which initiate stabilization of the tissue structure. Rapid and complete cross-linking is caused by the more slowly penetrating glutaraldehyde oligomers. This mixture is associated with the name Morris J. Karnovsky of Boston. It is an example of a great innovation that was published only in an unrefereed abstract (Karnovsky, 1965).
His original mixture contained 4% glutaraldehyde, which was a higher concentration than many people wanted to use (Hayat, 1981). Designations like “half-strength Karnovsky” became common parlance in the 1960s and 1970s. Primary fixative helped to resolve observations of tissues that were difficult to preserve em Electron microscopy . the introduction of Karnovsky fixative, and they are still commonly used.
Aldehydes are the most commonly used fixatives. They are used to stabilize the fine structural details of cells and tissues prior to light or electron microscopy.
Like glutaraldehyde , paraformaldehyde is also commonly used and will depolymerize into formalin when heated, making it an effective fixative too.
Why is glutaraldehyde used in electron microscopy?
Glutaraldehyde is one of the most frequently used fixatives. It reacts rapidly with proteins and, as a dialdehyde, stabilizes structures by cross-linking before there is any possibility of buffer extraction.
To avoid denaturing glutaraldehyde, it should be stored at 4°C.