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Thursday, April 18, 2013

Environmental Pollution: A Major Threat

Pollution is any, physical, chemical or biological changes that adversely affect the health, survival or activities of living organisms that alter the environment in an undesirable way.
Sources of Pollution

Sources of Pollution are of two types:

  • Natural Sources
  • Man made Sources

Natural Sources of Pollution:
Natural sources of pollution includes volcanic activity, algal blooms, dramatic changes in the ocean currents.
Volcanic activity can lead to alteration of climatic conditions. algal blooms can cause low level of Biological oxygen demand there by killing fishes. changes in the Ocean current can lead to poor nutrient cycling.

Man made sources
All human activities.
agricultural impact on environment.
Transportation of Hazardous materials
Incineration of waste products.
Industrial activities.
Pollutants are released accidentally, some times deliberately in a single step or continuously eg: effluents.leak or leaching of water.

Environmental Pollutants
Biological - Cholerae Bacterium, sewage pollution of water
Physical - Heat, Noise, Radioactivity, Particulate matter.
Chemical - chemicals are intrinsically harmful, eg: heavy metals, Xenobiotic compounds.
Most naturally occurring substances ie, pollutants whose increased concentration in environment can lead to health problems or environmental damage. Eg: chloride in water.
Priority Pollutants
US environmental protection agency has listed 129 priority pollutants which are dangerous and can cause health problems and environmental damage.

Persistent Organic Pollutants (POP's)
12 most notorious persistent organic pollutants (POPs) are called as dirty dozens: these are
1. DDT
2. Toxaphane
3. PCB's
4. Dioxins & Furans
5. Hexachloro Benzene
6. Chlordane
7. Dieldrin
8. Endrin
9. Aldrin
10. Mirex

Methanol Production

Methanol is synthetically produced from carbon dioxide and hydrogen.
CH4 + CO2 + H2O ---- 8H2 + 4CO
8H2 + 4CO ----- 4 CH3OH

Methanol from wood waste
Methanol can be produced from any carbonaceous material like coal, lignite, wood waste. Agricultural and garbage waste can also be used for methanol production. These raw materials needs additional processing steps to refine crude gas product to final synthetic gas product. The ratio is 2 Parts of H2 : 1 Part of CO.
The process is energy intensive and the yield is less.

Process Steps

  1. Partial Oxidation of wood waste (H2, CO, CO2, N2, etc.)
  2. Clean and Crude gas
  3. compress to 100 lb/inch sq./g
  4. Remove Carbon dioxide
  5. Remove residual carbon dioxide
  6. Remove Nitrogen and high carbon
  7. Compress tp 400lb/inchsq./g
  8. Shift gas into 2 parts of H2
  9. Compress to 2500 lb/inch sq./g
  10. Convert H2 + CO into Methanol
  11. Refine crude methanol to specification grade.

Tuesday, April 9, 2013

Sterilization - Autoclaving

An autoclave is a pressurized device designed to heat aqueous solutions above their boiling point to achieve sterilization. It was invented by Charles Chamberland in 1879. The term autoclave is also used to describe an industrial machine in which elevated temperature and pressure are used in processing materials.

Introduction to Autoclave:
Under ordinary circumstances (at standard pressure), liquid water cannot be heated above approximately 100 °C (99.99 °C at 101.325 kPa, 99.62 °C at 100 kPa) in an open vessel except for special situations. Further heating results in boiling, but does not raise the temperature of the liquid water. However, when water is heated in a sealed vessel such as an autoclave, it is possible to heat liquid water to a much higher temperature. As the container is heated the pressure rises due to the constant volume of the container (see the ideal gas law). The boiling point of the water is raised because the amount of energy needed to form steam against the higher pressure is increased.

A Lab Scale Autoclave
Lab scale autoclave

An Industrial Scale Autoclave

Uses of Autoclave:Autoclaves are widely used in microbiology, medicine, sterilizing instruments for body piercing, veterinary science, dentistry, podiatry and metallurgy. The large carbon-fiber composite parts for the Boeing 787, such as wing and fuselage parts, are cured in large autoclaves.

Principle of Autoclave
A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally. For example, when free flowing steam at a temperature of 100oC is placed under a pressure of 1 atmosphere above sea level pressure – that is, about 15 pounds of pressure per square inch (Psi) --- the temperature rises to 121oC. Increasing the pressure to 20 psi raises the temperature to 126oC. The relationship between temperature and pressure is shown in table 2. In this way steam is a gas, increasing its pressure in a closed system increases its temperature. As the water molecules in steam become more energized, their penetration increases substantially. This principle is used to reduce cooking time in the home pressure cooker and to reduce sterilizing time in the autoclave. It is important to note that the sterilizing agent is the moist heat, not the pressure.


It is essential to remove all the traps from the autoclave otherwise required temperature is not achieved(because air is bad conductor of heat) which leads to non sterility.

• Downward displacement (or gravity type) - As steam enters the chamber, it fills the upper areas as it is less dense than air. This compresses the air to the bottom, forcing it out through a drain.

• Steam pulsing - Some autoclaves remove air by using a series of steam pulses, in which the chamber is alternately pressurised and then depressurised to near atmospheric pressure.

• Vacuum pumps - Some autoclaves use vacuum pumps to suck air or air/steam mixtures from the chamber.

There are 3 sterilization cycles employed in an autoclave to sterilize different loads.

1. Standard cycle {for wrapped containers like glassware, test tubes, utensils}

2. Liquid cycle {for liquid loads like media and buffer in tightly sealed container}

3. HPHV [high pressure high vacuum] cycle {for fabric like lab coats, gloves, flexible tubes}

Limitations and Disadvantages of Autoclave:
Sterilization by autoclave method has certain limitations. For example, some plastic ware melts in the high heat, and sharp instruments often become dull. Moreover, many chemicals breakdown during the sterilization process and oily substances cannot be treated because they do not mix with water. Heat requires extra time to reach the center of solid materials, such as caned meats, because such materials do not develop the efficient heat-distributing convection currents that occur in liquids. Heating large containers also requires extra time. Unlike sterilizing aqueous solutions, sterilizing the surface of a solid requires that steam actually contact it.

Indicator of Sterilization Achievement:
Several commercially available methods can indicate whether sterilization has been achieved by heat treatment. Modern autoclaves have devices to maintain proper pressure and record internal temperature during operations. Regardless of the presence of such a device, the operator should check pressure periodically and maintain the appropriate pressure. Chemical reactions in which an indicator changes color when the proper times and temperatures have been reached. In some designs, the word "sterile" or "autoclaved" appears on wrappings or tapes. These tapes are not fully reliable because they do not indicate how long appropriate conditions were maintained. Tapes or other sterilization indicators should be placed inside and near the center of large packages of determine whether heat penetrated them. In another method, a pellet contained within a glass vial melts. A widely used test consists of preparations of specified species of bacterial endospores such as Bacillus stearothermophilus, impregnated into paper strips. The spore strip and an ampule of medium are enclosed in a soft plastic vial. The vial is placed in the center of the material to be sterilized and is autoclaved. After autoclaving, these can then be aseptically inoculated into culture media. Growth in the culture media indicates survival of the endospores and therefore inadequate processing. Other designs use endospore suspensions that can be released, after heating, into a surrounding culture medium within medium within the same vial.

Important Points to Remember For Autoclaving:
Steam under pressure fails to sterilize when the air is not completely exhausted. This can happen with the premature closing of autoclave's automatic ejector valve. The principles of heat sterilization have a direct bearing on home canning. To sterilize dry glassware, bandages, and the like, care must be taken to ensure that steam contacts all surfaces. For example, aluminum foil is impervious to steam and should not be used to wrap dry materials that are to be sterilized; paper should be used instead. Care should also be taken to avoid trapping air In the bottom of a dry container because trapped air will not be replaced by steam, which is lighter than air. The trapped air is the equivalent of a small hot-air oven, which, as we will see shortly, requires a higher temperature and longer time to sterilize materials. Containers that can trap air should be placed in a tipped position so that the steam will force out the air. Products that do not permit penetration by moisture, such as mineral oil or petroleum jelly, are not sterilized by the same methods that would sterilize aqueous solutions. This precaution is necessary because when an object is exposed to heat, its surface becomes hot much more quickly than its center.

Monday, April 8, 2013

Media & pH Optimization For E. Coli in Shake Flask

E. coli is Gram-negative, facultative anaerobic and non-sporulating bacteria. It can live on a wide variety of substrates. Optimal growth of E. coli occurs at 37°C, but some laboratory strains can multiply at temperatures of up to 49°C. Growth can be driven by aerobic or anaerobic respiration. Optimization of a particular parameter for any culture in any culture condition can be done by keeping all the other parameters constant. For optimization of the pH and the media for E. coli in shake flask conditions, all the other parameters such as temperature, shaker speed, media volume
per volume of flask, inoculum percentage etc should keep constant. E. coli grows well in neutral pH and hence media with pH 6.9 and 7.8 are used for optimization of the pH. LB broth, Nutrient broth and Tryptose soya broth are some best known media for E.coli and hence these are tried to optimize the media. The optimal pH and media are selected on the basis of the optical density given by the cultures at specific intervals.

List of Materials:

LB broth, Nutrient broth and Tryptose soya broth of pH 6.9 & 7.8, Conical flasks,E.coli culture, Test tubes, Test tube stand, Pipettes and Bunsen burner.

List of Equipments:

pH meter, vortex mixer, Spectrophotometer, Laminar air flow chamber and Shaker incubator.


Day 1: Media preparation and Inoculation

  1. The components of the media for 150 ml are weighed and dissolved in 100 ml and equally distributed into 2 conical flasks.
  2. The pH of the media in two flasks is adjusted to 6.9 and 7.8 respectively.
  3. The volume of media in each flask is made up to 75 ml with purified water.
  4. The flasks are autoclaved at 121°C, 15 lbs pressure for 30 minutes.
  5. The flasks are inoculated with 3 ml pre-inoculum of E.coli inside the laminar air flow chamber.
  6. The flasks are incubated at 37°C in the shaker incubator at 200 rpm.

Day 2: Observation

  1. 2 ml of the culture broth from both flasks are taken into separate test tubes and the pH and OD of the culture are checked at intervals of 16 hrs, 19 hrs, 20 hrs and 21 hrs after inoculation.

Observation & Inference

It was found that the LB broth of pH 6.9 is optimum for growth of E. coli in shake flask conditions from the above observations.

Production of Ethanol by Fermentation

Alcohol, chemically ethanol (C2H5OH) has been produced by fermentation for thousands of years.

Microbes used:
Certain yeasts and bacteria are employed for alcohol fermentation, the type of the organism chosen mostly depends on the nature of substrate used.In yeast Sacchromyces cervisea is widely used and in bacteria Zymomonas mobilis is most frequently used for alcohol fermentation.

Raw Materials used for Alcohol Fermentation
There are large number of raw materials which can serve as substrate for alcohol fermentation. They are broadly categorized as
  1. Sugary Raw Materials
  2. Starchy Raw Materials
  3. Cellulosic Raw Materials

Sugary raw materials includes Molases, whey, glucose, sucrose, etc.
Starchy raw materials includes Wheat, Rice, Maize, Potato.
Cellulosic raw materials includes Wood, agricultural wastes, etc.

Most of the raw materials of alcohol fermentation requires some degree of pretreatment. The actual process depends on the chemical composition of the raw materials.
Sugary raw materials require mild ot no pretreatment while cellulosic raw material need extensive pretreatment. This is because cellulosic substance have to be subjected to acidic or enzyme hydrolysis to release monosaccharide units that are needed for alcohol production.

Biosynthesis of Ethanol
ethanol production by fermentation

Glucose gets broken down to pyruvate by glycolysis under anaerobic conditions (in the absence of oxygen), pyruvate is converted to acetaldehyde by the enzyme pyruvate decarboxylase. Acetaldehyde is then reduced by alcohol dehydrogenase to form ethanol.

anaerobic fermentation

Regulation of Synthesis
Ethanol at high concentration in the medium inhibits its own biosynthesis, this is particularly observed when yeasts are fermentation organisms. it is generally seen that the growth of yeast ceases at 5% ethanol concentration. zymomonas mobilis can tolerate even higher alcohol concentration.

Production Process of Ethanol

Ethanol production, alcohol fermentation

Sunday, April 7, 2013

Automated Liquid Handling Systems

Liquid handling systems are instruments which helps in dispensing liquids to the specified locations. Automated liquid handling systems have robotic arm sensors which perfectly collect liquid and dispense it on the user specified location with precision. volume dispensed can be as low as to the level of nanolitres.

Automation has revolutionized and influenced the way scientists do research - maximizing accuracy, precision and throughput while minimizing time and consumable costs. However, the prevalent thought that automated systems are expensive, complicated, and space-consuming leave many scientists unwilling or unable to maximize the potential of their work.

There are many liquid handling systems are available in the market, each has got its own advantages and disadvantages.

Eppendorf's epmotion
epmotion, manufactured by eppendorf.


epMotion liquid handling system has various models like epMotion 5070, epMotion M5073, epMotion 5075 LH, epMotion 5075 TMX, etc.

epMotion 5075 Features
epMotion Liquid Handling system has the following characteristics
  • Automated Tool Exchange
  • 12 Decks or 12 work positions
  • Probe for sensing liquid level and the accessories
  • Ability to dispense small volumes.

Friday, April 5, 2013

qPCR Calculations

qPCR or Real time PCR  has got several applications in diagnostics, forensic science and in many other biotechnology areas. The main advantage of PCR is its sensitivity even very small quantities of DNA can be detected using this method making it a powerful tool in the diagnostics and other areas.
Lets us look into some of the problem which beginners face while preparing master mix and setting up qPCR reactions.

Master mix Preparation for qPCR
PCR reaction mix also known as master mix is a combination of Buffer, Primers, Probe, Taq Polymerase, etc. it is prepared as for the no of reactions to be done. it is prepared in bulk and aliquoted into individual PCR tubes or PCR strips.

There are commercially available master mixes of various strengths like 5X, 2X, etc (Eg: 2X Universal Master Mix from takara), for a 10uL PCR reaction 5uL of the 2X master mix is required. (required volume / concentration of the master mix =10/2= 5uL).

Primer - Probe Dilution:
Primer is an oligoncleotide which binds specifically  to its complementary sequence in the target DNA. Primers are designed in such a way that it will bind only to particular region of the target. Primers can be custom synthesized depending on the sequence of the target DNA to be detected.
Custom synthesized primers are usually shipped to the customer as lyophilized powder along with a data sheet. Data sheet will have the information about the synthesis scale, yield, concentration, Optical Density readings, volume required to make 100uM solution.

Primer Reconstitution & Dilution
Primer stocks are maintained at 100uM concentration and the working stock will be of 10uM (Varies depending on the convenience of the person working).
A 100 micro Molar (uM) primer stock solution corresponds to 100 picomoles/uL (pmols/uL). one of the doubt i had when i started working on real time PCR how 100uM = 100pmols/uL, yes it is lets see how it is:
before entering into that just look at the basic conversions:
as we all know 1 Molar solution has 1 mole/Litre (Recollect the defintion of Molarity,"Molarity is defined as the no of moles per liter of a solution"). Eg: 1M NaOH solution has 1 mole of NaOH in 1 Litre.
1micro Molar solution will have 1 micromole / litre of a solution.

1uM = 1micromole /Litre or 1*10^6 picomoles/ Litre
= 1*10^6 pmols/1000mL
=1*10^6/10^3 pmols/mL
=1*10^3 pmols/mL ; (1mL = 1000uL) so,
=1*10^3 pmols/1000uL
=1*10^3 pmols /10^3 uL
=1 pmol/uL.

Similarlly 100uM primer solution will have 100pmols/uL of primer.

On adding required volume of TE Buffer or Sterile water to the lyophilized powder as mentioned in the primer data sheet, 100uM primer solution will be obtained and this Procedure is called as Primer Reconstitution. Reconstituted primers can be stored at -20 dC.

Primer Analysis
Primer once reconstituted needs to be analyzed by taking OD@260 in a spectrophotometer for its concentration. even though the supplier provides the information in the data sheet, it is good to have the concentration checked before proceeding with the reaction. lets see how to check the primer concentration in a spectrophotometer.

Dilute the reconstituted primer 100 fold to a volume of 1mL. [10uL of primer stock + 990uL of TE Buffer].
read absorbance at OD260. using Oligo Calc primer concentration can be easily calculated by inputting the primer sequence and the OD reading.
[Note: multiply the OD 260 reading into 100, as we have diluted it 100 fold].

concentration of primer

Another method of checking the primer is by running an Oligonucleotide PAGE, (Oligonucleotide PAGE protocol). Primer degradation can be found out by running the oligonucleotide PAGE.

Primer Dilution: Preparation of primer working stock
Once primer is reconstituted, working stock need to be prepared for using in PCR reaction. (some people use directly from the primer stock), generally working stock is maintained as 10uM solution. a ten fold dilution of the stock solution can be done to get a 10uM primer working stock solution.
Lets say you need a 100uL of 10uM primer working stock;
C1*V1 = C2*V2
100*V1  = 10*100
V1= 10uL.

You need to take 10uL of the 100uM stock and add 90uL of water to obtain 100uL of 10uM solution.

Probe Reconstitution and Dilution
Probe reconstitution and dilution is also done in same way as mentioned above only difference is probes will be coluored while primers are colourless.
Probe is reconstituted to 100uM and diluted 10 times to get 10uM working stock.

Taq Polymerase
Taq polymerase is the enzyme which helps in the synthesis of DNA copies from the template. there are two types of Taq Polymerase available wild Taq and Hot Start Taq (More on Hotstart Taq Polymerase and Types).
Taq polymerase is available in various concentrations , lets see one and find out how it is used in PCR reaction.
Eg: Hot Start Taq Polymersae 5U/uL
you need 2units for your 10uL PCR reaction, how many uL of  taq to use:
here one thing you need to keep in mind the taq stock has a concentration of 5U/uL and you need 2 units/10uL. Make both the concentration to same units
2U/10uL = 0.2U/uL
so C1*V1 = C2*V2
5U/uL*V1 = 0.2U/uL * 10
= 0.4uL
you need to take 0.4uL of 5U/uL of Taq to get 2U/10uL.

PCR reaction volumes are made up to the required volume by using sterile water. quality of water is important in PCR. Distilled water or sterile water free of DNase and RNases are recommended for usage.

Template is your DNA / RNA sample.

Oil, mineral oil is added to the PCR reaction tube inorder to prevent evaporation. it acts as a vapour barrier.In some of the mordern PCR machines oil addition to the PCR tube is not needed.

A general PCR Reaction compostition of 10uL PCR Reaction

2X Buffer                   - 5.0uL
10uM FOrward Primer - 0.3uL
10uM Reverse Primer - 0.3uL
Probe                          -  0.2uL
Water                        - 0.2uL
Template            - 4.0uL
Total                         - 10.0uL