- Overview
- Wet preparation equipment
- Microscopy
- Spectroscopy
- Chromatography (Separation)
- Chromatography (detection)
- Molecular Biology
- Cell culture
- Electrophysiology
- 3D Bioprinting
- Various methods
Biological research sooner or later leads to investigations on "living stuff". This can mean pure observation in nature, whole organism (multicellular) experiments in the lab, but with the development of molecular and growing techniques, also cell and tissue cultivation. Experimentation, especially on animals, where pain or suffering can be introduced, goes hand in hand with ethics and morality and it is essential to minimize the impact made on the experimental specimen. This is often regarded to as the rule of three Rs - reduction, replacement and refinement. Cell and tissue culture is an essential tool, not only to achieve this goal, but also to make experiments which would otherwise be impossible. Unless some very specific cell type is to be investigated, the cells typically do not require to be extracted separately, since standardized cell lines are already available all over the globe. To work with or modify these, however, a laboratory requires special registration and allowances.
Some cells (especially animal cells) are very sensitive to environmental changes. To keep them well and healthy for cultivation and experimentation, everything needs to be just right. For example, liquids which are exchanged (growth medium) od added to the culture (enzymes or other molecules), need to have the same temperature as the incubated cells. Therefore, they are usually heated to the right temperature in a water bath. This is actually very straight forward, however, the temperature has to be just right, so it does not shock the cells, but also cannot be overcooked, since some of the contained molecules are sensitive to heat.
Keywords: warm, water, bath
Prices for warm water baths usually vary between 500 and 1000€, depending on the size, precision, etc. An alternative to laboratory warm water baths are sous-vide cookers (slow cookers), which are available for less, but often show the same capabilities.
Interestingly, the existing "lab-grade" DIY warm water baths, mostly seem to come as adaptations of DIY slow cookers, such as this one. Two designs seem to do their job very well, which can be found here and here.
The existing models seem to be capable of setting and holding a constant temperature (+/- 0.5°C), which is good enough.
####5. Requirements and plans Warm water baths need to finely adjust and hold the temperature of the contained water, which heats other objects inside. For the water to have an even temperature distribution it has to be continuously mixed.
Source: By William Rafti of the William Rafti Institute, Attribution, https://commons.wikimedia.org/w/index.php?curid=18424292
When working with cells, tissues, the handling equipment, as well as the culture media, need to be sterile. There are many types of sterilization, using irradiation (such as UV, X-ray or gamma radiation), chemical agents (NO₂,Ethylene oxide,..), but most common in biological laboratories is sterilization with hot steam by autoclaving (121°C in a 100% steam atmosphere at 1.04 bar gauge pressure for 5 minutes).
Three types of steam sterilisation cycle are commonly required: growth media (and other liquids); waste (e.g. biohazard bags); solid objects (e.g. flasks, bottles, pipette tips, etc). For growth media and liquid sterilisation the liquid itself must reach 121°C. Since trapped air pockets are not a concern, steam can simply be allowed to passively displace the air in the chamber (class N cycle). For waste and solid object sterilisation it is necessary for steam to completely penetrate all areas of the autoclave load. This is often achieved with a pulsed vacuum system (class B cycle) which removes air pockets in a load containing wrapped objects, and objects such as pipette tips which are prone to causing air pockets.
Keywords: autoclave, moist, heat, steam, sterilization
Commercial autoclaves with large sterilisation chambers precise programmable settings (temperature, pressure, humidity) cost a few thousand €, those additionally capable of class B sterilisation cost even more.
The use of pressure cookers is established amongst DIY biologists and works well as a cheap and easy solution for sterilising liquids and growth media. Most pressure cooker designs have the pressure vent located at the top of the chamber which means that the steam (lighter than air) will never fully displace the air in the chamber. If a pressure cooker with a vent located at the bottom can be found this will be far superior to the standard design, however, a raised shelf inside the chamber can be added to give better confidence when sterilising non-hollow solids.
Two of the more advanced (and also not very cheap) can be found here and here. Due to its large size, the last one is even more expensive than used small commercial ones. This is, however, largely dependent on the needs one has for sterilization.
Using a simple pressure cooker for sterilization will suffice in many instances. For labs requiring autoclaves capable of sterilising solid waste to a high level of certainty (e.g. labs dealing with genetically modified microbes in the EU) pressure cookers may not suffice and an autoclave capable of class B cycles should be sought.
The existing DIY devices are promising, however made for specific purposes and not generally required.
A class N sterilisation capable autoclave should have the capabilities of preadjusted temperature and pressure regulation, which is controlled by a feedback loop and a little electronics. If possible the pressure vent should be located at the base of the chamber so that steam is able to fully displace the air in the chamber. A self-regulating system based on a pressure cooker should be simple and cheap enough for the needs of most DIY biologists and enable them to sterilize more sensitive material.
Source: By No machine-readable author provided. Own work assumed (based on copyright claims)., Public Domain, https://commons.wikimedia.org/w/index.php?curid=609576
Laminar flow hoods and clean benches come in two major categories: those designed to isolate samples from the environment but not the environment from the samples; and those designed to isolate the samples from the environment and the environment from the samples (often referred to as "class II safety cabinets"). Before air enters the chamber, it is filtered with a HEPA filter (highly efficient particle filter, which removes almost 100% of particles >0.3µm). Then it is directed in a laminar fashion through the interior of the chamber and outward. The exhaust is either in the front (where the experimenter works), which is potentially dangerous, or travels through a porous mesh (work surface) and leaves the chamber on the same side as it enters, possibly going through an additional HEPA filter to remove any sample particulate before the air returns to the environment.
Keywords: laminar, flow, hood, cabinet, clean, bench
The prices of commercial laminar flow hoods vary, depending on their size, protection standard, etc. and can range from around €1000 to several thousand euros.
With increasing marijuana and especially fungi cultivation in the last years, the DIY equipment for sterile work seems to have developed quite a lot. Laminar flow hoods are a good example of this. Currently many DIY designs of non-class-II-type hoods are available, using different airflow directions (horizontal: from back to front - the majority of DIY designs, or vertical: from top to bottom). Most designs are fairly simple. A few examples are: a portable laminar flow hood, or this somewhat more advanced design with a prefilter, both horizontal flow designs. Vertical flow designs include the simple fresh cap DIY laminar flow hood and the design from the London Biohackspace. There is also a guide on calculating and determining the appropriate chamber size in regard to the blower and filter sizes, which can be found here.
However, the cheapest, easiest to set up, method of creating a sterile work area is to use a a bunsen burner or small camping gas burner to create an updraft in the area surrounding the burner. The sterile zone around a burner may be less reliable than a flow hood workbench, however for the purposes of many procedures such a set up is sufficient as very low amounts of contamination can be achieved if the work is carried out carefully.
Reducing the laminar flow hood to its parts (which really aren't so many) and assembling one by oneself, seems to drastically reduce costs and the example from the London Biohackspace seems to achieve aseptic conditions. Thus we can assume that the DIY systems are both good enough and reasonable. In many cases however, it is far cheaper and more practical to simply use a burner setup.
It is crucial to note that all the DIY designs linked above are not suitable for protecting an experimenter from the samples being handled. For working with unknown microbes or anything potentially pathogenic there is no safe replacement to commercially constructed and tested class II safety cabinets.
Laminar flow hoods are meant to protect the samples inside from particulate impurities, bacteria and fungi, which is accomplished by filtering the air, that enters the chamber. Since the chamber is also open on the side of the experimenter, any major turbulences in airflow could lead to the impurities entering from there. Therefore the airflow should be relatively slow (~0.5 m/s) and as laminar as possible. It is also beneficial to make the chamber from materials which are easy to clean and less likely to harbor growth of micro organisms.
Source: http://www.thermoscientific.com/en/community/growth-passage.html
The function of an incubator seems very straightforward. It typically consists of a chamber used for the incubation of things, in molecular and cell biology mostly for cells, tissues and microorganisms. Many of these are very sensitive about their environmental conditions, which include chemical properties of their growth medium, but also temperature and ambient gas composition. The latter two can be adjusted in an incubator.
Keywords: incubator, incubation, chamber, cultivation
Incubators come in different froms and sizes and as a consequence - in different prices as well. They range from small egg (bird or reptile) warmers, to professional laboratory devices with an almost sterile environment, precise temperature, shaking speed, and gas (CO₂) control etc. The latter kind is usually very expensive, costing several thousand €. Smaller, simple devices are available for a few hundred, or even below €100 (hobby/terrarist grade).
Typically, DIY incubators consist of a styrofoam chamber (with a door), heat source, temperature sensor and a regulation circuit, as shown in this video. A similar build was made by Biodesign, where also a compact temperature controller was developed - Fermento. Such designs can be combined with orbital shakers to create a device capable of incubating cell culture under shaking conditions. For more elaborate culturing protocols, e.g. mammalian cell culture, a more advanced device was devised at the Pelling Lab, where a DIY CO₂ incubator was developed.
Whether it is worthwhile to build a DIY incubator, very much depends on the characteristics you require. For simple nonsterile incubation at a constant temperature, cheap commercial devices seem a better choice (e.g. large chicken egg incubators can be bought for €70). For sensitive eukaryotic cell line incubation, this is another matter completely! For such research, a DIY design is feasible, if you can achieve the neccessary conditions, e.g. as it was done in the Pelling lab.
An incubator for bacterial cell culture has few requirements: ability to set a stable temperature between 25°C-37°C and optionally the incorporation of a variable speed shaking platform to speed up culturing times.
An incubator suitable for eukaryotic cell cultivation requires a chamber that is easily cleanable, with an airtightly sealable door/opening, precise temperature control, that can be adjusted and held constant and CO₂ regulation circuit that can sustain a 5% concentration.