DIYbio/FAQ/Methods

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DIYbio FAQ v1.5: "The biohacker's FAQ"

This FAQ for DIYbio is actively maintained by it's editors, and by you! Edit your contributions directly or email updates to the DIYbio email list, diybio@googlegroups.com.
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This topic Methods is part of the DIYBio FAQ

Please update this FAQ mercilessly with Q&A !

DIYbio Safety Manual

The DIYbio safety manual is in early stages, refer to it here: [DIYbio Safety Manual 1.0]


Laboratory Basics

The following information is for basic lab technique.


USING PHYSICAL AGENTS TO CONTROL MICROORGANISMS

This section is quoted from BIOL 230 MICROBIOLOGY LABORATORY MANUAL by Dr. G.E. Kaiser, Copyright © Gary E. Kaiser, Updated: March 4, 1999


A. INTRODUCTION TO THE CONTROL OF MICROORGANISMS

Control of microorganisms is essential in order to prevent the transmission of diseases and infection, stop decomposition and spoilage, and prevent unwanted microbial contamination.

Microorganisms are controlled by means of physical agents and chemical agents. Physical agents include such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration. Control by chemical agents refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals.

Basic terms used in discussing the control of microorganisms include:

1. Sterilization Sterilization is the process of destroying all living organisms and viruses. A sterile object is one free of all life forms, including bacterial endospores, as well as viruses.

2. Disinfection Disinfection is the elimination of microorganisms from inanimate objects or surfaces.

3. Decontamination Decontamination is the treatment of an object or inanimate surface to make it safe to handle.\

3. Disinfectant A disinfectant is an agents used to disinfect inanimate objects but generally to toxic to use on human tissues.

4. Antiseptic An antiseptic is an agent that kills or inhibits growth of microbes but is safe to use on human tissue.

6. Sanitizer A sanitizer is an agent that reduces, but may not eliminate, microbial numbers to a safe level.

5. Antibiotic An antibiotic is a metabolic product produced by one microorganism that inhibits or kills other microorganisms.

6. Chemotherapeutic antimicrobial chemical Chemotherapeutic antimicrobial chemicals are synthetic chemicals that can be used therapeutically.

7. Cidal An agent that is cidal in action will kill microorganisms and viruses.

8. Static An agent that is static in action will inhibit the growth of microorganisms.

B. TEMPERATURE

Microorganisms have a minimum, an optimum, and a maximum temperature for growth. Temperatures below the minimum usually have a static action on microorganisms. They inhibit microbial growth by slowing down metabolism but do not necessarily kill the organism. Temperatures above the maximum usually have a cidal action, since they denature microbial enzymes and other proteins. Temperature is a very common and effective way of controlling microorganisms.

1. High Temperature

Vegetative microorganisms can generally be killed at temperatures from 50°C to 70°C with moist heat. Bacterial endospores, however, are very resistant to heat and extended exposure to much higher temperature is necessary for their destruction. High temperature may be applied as either moist heat or dry heat.

a. Moist heat

Moist heat is generally more effective than dry heat for killing microorganisms because of its ability to penetrate microbial cells. Moist heat kills microorganisms by denaturing their proteins (causes proteins and enzymes to lose their three-dimensional functional shape). It also may melt lipids in cytoplasmic membranes.

1. Autoclaving

Autoclaving employs steam under pressure. Water normally boils at 100°C; however, when put under pressure, water boils at a higher temperature. During autoclaving, the materials to be sterilized are placed under 15 pounds per square inch of pressure in a pressure-cooker type of apparatus. When placed under 15 pounds of pressure, the boiling point of water is raised to 121°C, a temperature sufficient to kill bacterial endospores.

The time the material is left in the autoclave varies with the nature and amount of material being sterilized. Given sufficient time (generally 15-45 minutes), autoclaving is cidal for both vegetative organisms and endospores, and is the most common method of sterilization for materials not damaged by heat.

2. Boiling water

Boiling water (100°C) will generally kill vegetative cells after about 10 minutes of exposure. However, certain viruses, such as the hepatitis viruses, may survive exposure to boiling water for up to 30 minutes, and endospores of certain Clostridium and Bacillus species may survive even hours of boiling.

b. Dry heat

Dry heat kills microorganisms through a process of protein oxidation rather than protein coagulation. Examples of dry heat include:

1. Hot air sterilization

Microbiological ovens employ very high dry temperatures: 171°C for 1 hour; 160°C for 2 hours or longer; or 121°C for 16 hours or longer depending on the volume. They are generally used only for sterilizing glassware, metal instruments, and other inert materials like oils and powders that are not damaged by excessive temperature.

2. Incineration

Incinerators are used to destroy disposable or expendable materials by burning. We also sterilize our inoculating loops by incineration.

c. Pasteurization

Pasteurization is the mild heating of milk and other materials to kill particular spoilage organisms or pathogens. It does not, however, kill all organisms. Milk is usually pasteurized by heating to 71.6°C for at least 15 seconds in the flash method or 62.9°C for 30 minutes in the holding method.

2. Low Temperature

Low temperature inhibits microbial growth by slowing down microbial metabolism. Examples include refrigeration and freezing. Refrigeration at 5°C slows the growth of microorganisms and keeps food fresh for a few days. Freezing at -10°C stops microbial growth, but generally does not kill microorganisms, and keeps food fresh for several months.


C. DESICCATION

Desiccation, or drying, generally has a static effect on microorganisms. Lack of water inhibits the action of microbial enzymes. Dehydrated and freeze-dried foods, for example, do not require refrigeration because the absence of water inhibits microbial growth.

D. OSMOTIC PRESSURE

Microorganisms, in their natural environments, are constantly faced with alterations in osmotic pressure. Water tends to flow through semipermeable membranes, such as the cytoplasmic membrane of microorganisms, towards the side with a higher concentration of dissolved materials (solute). In other words, water moves from greater water (lower solute) concentration to lesser water (greater solute) concentration.

When the concentration of dissolved materials or solute is higher inside the cell than it is outside, the cell is said to be in a hypotonic environment and water will flow into the cell (Fig. 1). The rigid cell walls of bacteria and fungi, however, prevent bursting or plasmoptysis. If the concentration of solute is the same both inside and outside the cell, the cell is said to be in an isotonic environment (Fig. 2). Water flows equally in and out of the cell. Hypotonic and isotonic environments are not usually harmful to microorganisms. However, if the concentration of dissolved materials or solute is higher outside of the cell than inside, then the cell is in a hypertonic environment (Fig. 3). Under this condition, water flows out of the cell, resulting in shrinkage of the cytoplasmic membrane or plasmolysis. Under such conditions, the cell becomes dehydrated and its growth is inhibited.

The canning of jams or preserves with a high sugar concentration inhibits bacterial growth through hypertonicity. The same effect is obtained by salt-curing meats or placing foods in a salt brine. This static action of osmotic pressure thus prevents bacterial decomposition of the food. Molds, on the other hand, are more tolerant of hypertonicity. Foods, such as those mentioned above, tend to become overgrown with molds unless they are first sealed to exclude oxygen. (Molds are aerobic.)

E. RADIATION

1. Ultraviolet Radiation

The ultraviolet portion of the light spectrum includes all radiations with wavelengths from 100 nm to 400 nm. It has low wave-length and low energy. The microbicidal activity of ultraviolet (UV) light depends on the length of exposure: the longer the exposure the greater the cidal activity. It also depends on the wavelength of UV used. The most cidal wavelengths of UV light lie in the 260 nm - 270 nm range where it is absorbed by nucleic acid.

In terms of its mode of action, UV light is absorbed by microbial DNA and causes adjacent thymine bases on the same DNA strand to covalently bond together, forming what are called thymine-thymine dimers (see Fig. 4). As the DNA replicates, nucleotides do not complementary base pair with the thymine dimers and this terminates the replication of that DNA strand. However, most of the damage from UV radiation actually comes from the cell trying to repair the damage to the DNA by a process called SOS repair. In very heavily damaged DNA containing large numbers of thymine dimers, a process called SOS repair is activated as kind of a last ditch effort to repair the DNA. In this process, a gene product of the SOS system binds to DNA polymerase allowing it to synthesize new DNA across the damaged DNA. However, this altered DNA polymerase loses its proofreading ability resulting in the synthesis of DNA that itself now contains many misincorporated bases. In other words, UV radiation causes mutation and can lead to faulty protein synthesis. With sufficient mutation, bacterial metabolism is blocked and the organism dies. Agents such as UV radiation that cause high rates of mutation are called mutagens.

The effect of this inproper base pairing may be reversed to some extent by exposing the bacteria to strong visible light immediately after exposure to the UV light. The visible light activates an enzyme that breaks the bond that joins the thymine bases, thus enabling correct complementary base pairing to again take place. This process is called photoreactivation.

UV lights are frequently used to reduce the microbial populations in hospital operating rooms and sinks, aseptic filling rooms of pharmaceutical companies, in microbiological hoods, and in the processing equipment used by the food and dairy industries.

An important consideration when using UV light is that it has very poor penetrating power. Only microorganisms on the surface of a material that are exposed directly to the radiation are susceptible to destruction. UV light can also damage the eyes, cause burns, and cause mutation in cells of the skin.

2. Ionizing Radiation

Ionizing radiation, such as X-rays and gamma rays, has much more energy and penetrating power than ultraviolet radiation. It ionizes water and other molecules to form radicals (molecular fragments with unpaired electrons) that can disrupt DNA molecules and proteins. It is often used to sterilize pharmaceuticals and disposable medical supplies such as syringes, surgical gloves, catheters, sutures, and petri plates. It can also be used to retard spoilage in seafoods, meats, poultry, and fruits.


USING DISINFECTANTS ANTISEPTICS AND SANITIZERS TO CONTROL MICROORGANISMS

This section is quoted from BIOL 230 MICROBIOLOGY LABORATORY MANUAL by Dr. G.E. Kaiser, Copyright © Gary E. Kaiser, Updated: March 4, 1999


A. DISINFECTANTS, ANTISEPTICS, AND SANITIZERS

Disinfection is the elimination of microorganisms from inanimate objects or surfaces, whereas decontamination is the treatment of an object or inanimate surface to make it safe to handle.

a. The term disinfectant is used for an agent used to disinfect inanimate objects or surfaces but is generally to toxic to use on human tissues.

b. The term antiseptic refers to an agent that kills or inhibits growth of microbes but is safe to use on human tissue.

c. The term sanitizer describes an agent that reduces, but may not eliminate, microbial numbers to a safe level.

Because disinfectants and antiseptics often work slowly on some viruses - such as the hepatitis viruses, bacteria with an acid-fast cell wall such as Mycobacterium tuberculosis, and especially bacterial endospores, produced by the genus Bacillus and the genus Clostridium, they are usually unreliable for sterilization - the destruction of all life forms.

There are a number of factors which influence the antimicrobial action of disinfectants and antiseptics, including:

1. The concentration of the chemical agent.

2. The temperature at which the agent is being used. Generally, the lower the temperature, the longer it takes to disinfect or decontaminate.

3. The kinds of microorganisms present. Endospore producers such as Bacillus species, Clostridium species, and acid-fast bacteria like Mycobacterium tuberculosis are harder to eliminate.

4. The number of microorganisms present. The more microorganisms present, the harder it is to disinfect or decontaminate.

5. The nature of the material bearing the microorganisms. Organic material such as dirt and excreta interferes with some agents.

The best results are generally obtained when the initial microbial numbers are low and when the surface to be disinfected is clean and free of possible interfering substances.

There are 2 common antimicrobial modes of action for disinfectants, antiseptics, and sanitizers:

1. They may damage the lipids and/or proteins of the semipermeable cytoplasmic membrane of microorganisms resulting in leakage of cellular materials needed to sustain life.

2. They may denature microbial enzymes and other proteins, usually by disrupting the hydrogen and disulfide bonds that give the protein its three-dimensional functional shape. This blocks metabolism.

A large number of such chemical agents are in common use. Some of the more common groups are listed below:

1. Phenol and phenol derivatives

Phenol (5-10%) was the first disinfectant commonly used. However, because of its toxicity and odor, phenol derivatives are now generally used. These include orthophenylphenol, hexachlorophene, triclosan, hexylresorcinol, and chlorhexidine. Orthophenylphenol is the agent in Lysol®, O-syl®, Staphene®, and Amphyl®. Hexachlorophene in a 3% solution is combined with detergent and is found in PhisoHex®. Triclosan is a chlorine-containing phenolic antiseptic very common in antimicrobial soaps and other products. Hexylresorcinol is in throat lozenges and ST-37. A 4% solution of chlorhexidine in isopropyl alcohol and combined with detergent (Hibiclens® and Hibitane®) is a common handwashing agent and surgical handscrub. These agents kill most bacteria, most fungi, and some viruses, but are usually ineffective against endospores. They alter membrane permeability and denature proteins.

2. Soaps and detergents

Soaps are only mildly microbicidal. Their use aids in the mechanical removal of microorganisms by breaking up the oily film on the skin (emulsification) and reducing the surface tension of water so it spreads and penetrates more readily. Some cosmetic soaps contain added antiseptics to increase antimicrobial activity.

Detergents may be anionic or cationic. Anionic (negatively charged) detergents, such as laundry powders, mechanically remove microorganisms and other materials but are not very microbicidal. Cationic (positively charged) detergents alter membrane permeability and denature proteins. They are effective against many vegetative bacteria, some fungi, and some viruses. However, bacterial endospores and certain bacteria such as Mycobacterium tuberculosis and Pseudomonas species are usually resistant. They are also inactivated by soaps and organic materials like excreta. Cationic detergents include the quaternary ammonium compounds such as benzalkonium chloride, zephiran, diaprene, roccal, ceepryn, and phemerol.

3. Alcohols

70% solutions of ethyl or isopropyl alcohol are effective in killing vegetative bacteria, enveloped viruses, and fungi. However, they are usually ineffective against endospores and non-enveloped viruses. Once they evaporate, their cidal activity will cease. Alcohols denature membranes and are often combined with other disinfectants, such as iodine, mercurials, and cationic detergents for increased effectiveness.

4. Acids and alkalies

Acids and alkalies alter membrane permeability and denature proteins and other molecules. Salts of organic acids, such as calcium propionate, potassium sorbate, and methylparaben, are commonly used as food preservatives. Undecylenic acid (Desenex®) is used for dermatophyte infections of the skin. An example of an alkali is lye (sodium hydroxide).

5. Heavy metals

Heavy metals, such as mercury, silver, and copper, denature proteins. Mercury compounds (mercurochrome, metaphen, merthiolate) are only bacteriostatic and are not effective against endospores. Silver nitrate (1%) is sometimes put in the eyes of newborns to prevent gonococcal ophthalmia. Copper sulfate is used to combat fungal diseases of plants and is also a common algicide. Selinium sulfide kills fungi and their spores.

6. Chlorine

Chlorine gas reacts with water to form hypochlorite ions, which in turn denature microbial enzymes. Chlorine is used in the chlorination of drinking water, swimming pools, and sewage. Sodium hypochlorite is the active agent in household bleach. Calcium hypochlorite, sodium hypochlorite, and chloramines (chlorine plus ammonia) are used to sanitize glassware, eating utensils, dairy and food processing equipment, hemodialysis systems, and treating water supplies.

7. Iodine and iodophores

Iodine also denatures microbial proteins. Iodine tincture contasns a 2% solution of iodine and sodium iodide in 70% alcohole. Aqueous iodine solutions containing 2% iodine and 2.4% sodium iodide are commonly used as a topical antiseptic. Iodophores are a combination of iodine and an inert polymers such as polyvinylpyrrolidone that reduces surface tension and slowly releases the iodine. Iodophores are less irritating than iodine and do not stain. They are generally effective against vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses, and some endospores. Examples include Wescodyne®, Ioprep®, Ioclide®, Betadine®, and Isodine®.

8. Aldehydes Aldehydes, such as formaldehyde and glutaraldehyde, denature microbial proteins. Formalin (37% aqueous solution of formaldehyde gas) is extremely active and kills most forms of microbial life. It is used in embalming, preserving biological specimens, and in preparing vaccines. Alkaline glutaraldehyde (Cidex®), acid glutaraldehyde (Sonacide®), and glutaraldehyde phenate solutions (Sporocidin®) kill vegetative bacteria in 10-30 minutes and endospores in about 4 hours. A 10 hour exposure to a 2% glutaraldehyde solution can be used for cold sterilization of materials.

9. Ethylene oxide gas

Ethylene oxide is one of the very few chemicals that can be relied upon for sterilization (after 4-12 hours exposure). Since it is explosive, it is usually mixed with inert gases such as freon or carbon dioxide. Gaseous chemosterilizers, using ethylene oxide, are commonly used to sterilize heat-sensitive items such as plastic syringes, petri plates, textiles, sutures, artificial heart valves, heart-lung machines, and mattresses. Ethylene oxide has very high penetrating power and denatures microbial proteins. Vapors are toxic to the skin, eyes, and mucous membranes and are also carcinogenic. Another gas that is used as a sterilant is chlorine dioxide which denatures proteins in vegetative bacteria, bacterial endospores, viruses, and fungi.


B. EVALUATION OF DISINFECTANTS, ANTISEPTICS, AND SANITIZERS

It is possible to evaluate disinfectants, antiseptics, and sanitizers using either in vitro or in vivo tests. An in vitro test is one done under artificial, controlled laboratory conditions. An in vivo test is one done under the actual conditions of normal use.


C. EFFECTIVENESS OF HAND WASHING

There are 2 categories of microorganisms, or flora, normally found on the hands. Resident flora are the normal flora of the skin. Transient flora are the microorganisms you pick up from what you have been handling. It is routine practice to wash the hands prior to and after examining a patient and to do a complete regimented surgical scrub prior to going into the operating room. This is done in order to remove the potentially harmful transient flora, reduce the number of resident flora, and disinfect the skin.

Actual sterilization of the hands is not possible since microorganisms live not only on the surface of the skin but also in deeper skin layers, in ducts of sweat glands, and around hair follicles. These normal flora are mainly nonpathogenic staphylococci (Lab 15) and diphtheroid bacilli.

USING ANIMALS FOR EXPERIMENTATION

""In addition to the minimum legalities of working with animals, there are additional social and ethical considerations and guidelines, analyzing things such as the necessity of the research, protection of the animal research subjects against suffering, etc. If the individual / group of individuals / non-profit organization already has an institutional animal research committee, they are the best ones to talk to. If not, I'd suggest considering collaborating with an institution with established practices, eg a university or CRO, or redesigning the study to avoid the use of vertebrate animals. ""

-- ridgway on DIYbio google group

USING ANIMALS

Animals must be handled according to proper methods. Many institutions require animal researchers to be certified through a training program which informs them about institutional policies and procedures. Generally speaking, no license is needed or even available.

If you want to know the specifics on care in the US, please refer to the following:


""Overall: Don't do this at home. Please. Stick with microorganisms and insects. Please. For the sake of the animals, for the sake of your own health, and for the sake of your research. The more complicated your system gets the noisier it gets (in terms of data). If you want to do animal studies, please get the proper training. To the EU/US discussion: I have worked with lab animals in the US for quite some time. We do get licensed, but it's not a state thing, it's done at each institution. Go to a new institution and you have to get licensed all over again, because each institution is different. There is a difference in philosophy of how animal care is managed between the EU and the US. As I understand it, the EU takes an engineering approach; A cage of certain dimensions can have no more than so many animals, bedding must be of a specific composition and changed with a certain frequency, the facility must have this laundry list of specifications, etc. The US system is a performance standard. This means that stress is minimized, and it's more of a judgement call. Cage sizes and such are suggested (by NRC1996 & the Animal Welfare Act), but not mandated. Nobody shows up with a measuring tape and calculates square centimeters/animal, they just show up and say "That's too crowded." There are pros & cons to each. The classic example is in nonhuman primates. Two monkeys of the same species will have different requirements; one may be highly social, another may thrive more when left alone. You can't give a rigid guideline that controls for both. Same for mice. I use inbred C57's, have for years; they're basically clones of each other, but I still have mice that simple do better if in a cage alone and other mice that are obviously stressed unless they are with at least 2-3 other mice. I had one knockout strain that unless housed communally upon weaning, they would stop eating and probably starve to death/dehydrate if I had let them. But after 6 months of age, two males in the same cage would fight to the death. Two females in the same cage would strip each other of their fur in a single night. It was just something you had to watch, and the flexibility of the performance based system made it possible for me to do it and not technically be in violation. So who makes the call? From day to day, it's the Institutional Animal Care and Use Committee (IACUC). They oversee lab animal management departments, they approve protocols, and will yank facilities rights from researchers that don't adhere. IACUC is in turn watched over by AAALAC, a private nonprofit. AAALAC inspects every few years and comes up with any changes that need to be made. If they are not addressed, you can lose accreditation. If you lose accreditation, you pretty much aren't going to do any animal research any more. NIH won't fund you, and vendors won't sell to you.""

-- sgt york, DIYbio google group

This post highlights many misconceptions about animal research in the US, and presents a discouraging picture. In reality, AAALAC accreditation is NOT required for animal research in the US, nor is it required for NIH grants. Additionally, you cannot obtain AAALAC accreditation unless you already have an animal research facility in operation with your animal research area "mostly filled". You must also have an IACUC. For these reasons it is impractical for individuals or small organizations to obtain AAALAC accreditation.

DOSAGE FOR ANIMALS

A few helpful sites concerning what is typically used in labs and the recommended dosages:

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