Archive for April, 2014

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Clean Room – Its Applications and Control of Contaminants

April 23rd, 2014

Clean rooms are defined as a specially constructed enclosed area, environmentally controlled with respect to airborne particulates, temperature, humidity, air pressure, air flow patterns, air motion, vibration, noise, viable (living) organisms, and lighting.

Particulate control includes:

  • Particulate and microbial contamination
  • Particulate concentration and dispersion

Particulate/Contaminants must be continually removed from the air. The level to which these particles need to be removed depends upon the standards required. These standards are

  • “Federal Standard 209E” defines a clean room as a room in which the concentration of airborne particles is controlled to specified limits.
  • “British Standard 5295”defines a clean room as a room with control of particulate contamination, constructed and used in such a way as to minimize the introduction, generation and retention of particles inside the room and in which the temperature, humidity, airflow patterns, air motion and pressure are controlled.

Airborne particles occur in nature as dust, pollen, bacteria, miscellaneous living and dead organisms, and sea spray. Industry generates particles from combustion processes, chemical vapors, and friction in manufacturing equipment. People in the workspace generate particles in the form of skin flakes, lint, cosmetics, and respiratory emissions. All these particulates are either to be eliminated, diluted or prevented from settling on to the product surfaces.

The purpose of the clean room air-conditioning system is to supply airflow in sufficient volume and cleanliness to support the cleanliness rating of the room. Air is introduced into the clean room in a manner to prevent stagnant areas where particles could accumulate. The air must also be conditioned to meet the clean room temperature and humidity requirements. In addition, enough conditioned makeup air must be introduced to maintain the specified positive pressurization.
HEPA filters are a critical component in clean rooms. Clean room environments require highly filtered air that is frequently changed and delivered at precise conditions. Air-Handling units for clean room application require specific custom units that accommodate laminar air flow, HEPA and ULPA filtration, and sealed-insulation construction.

Applications of Clean Rooms       
Clean rooms are typically used in manufacturing, packaging and research facilities associated with the following industries

  • Semiconductor ; this industry drives the state of the art clean room design and this industry accounts for a significant number of all operating clean rooms.
  • Pharmaceutical ; clean rooms control living particles that would produce undesirable bacterial growth in the preparation of biological, pharmaceutical and other medical products as well as in genetic engineering research.
  • Aerospace; the manufacturing and assembling of aerospace electronics, missiles and satellites were the first application of clean rooms. Large volume clean room spaces with extreme cleanliness are involved.
  • Miscellaneous applications; other uses include advanced materials research, laser and optic industries, microelectronic facility, paint room and in some aseptic foods production. Also in some high infection risk areas of hospitals.

Control of contaminants

Control is primarily through airflow design. Construction finishes; personnel and garments; materials and equipments are sources of particulate contaminations that must be controlled. Important control precautions include:

  • Walls, floors, ceiling tiles, lighting fixtures, doors and windows are construction materials that must be carefully selected to meet clean room standards.
  • People must wear garments to minimize the release of particles into the space. The type of garments depends on the level of cleanliness required by a process. Smocks, coveralls, gloves and head and shoe cover are clothing accessories commonly used in clean spaces.
  • Materials and equipments must be cleaned before entering the clean room.
  • Room entrances such as sir locks and pass through are used to maintain pressure differentials and reduce contaminants.

Air shower is used to remove contaminants from personnel before entering the clean spaces.

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CENTRIFUGE

April 22nd, 2014

Centrifugation is a technique of separation of mixtures e.g. subcellular organelles that involves the centrifugal force using a CENTRIFUGE(A centrifuge uses centrifugal force to isolate suspended particles from their surrounding medium on either a batch or a continuous-flow basis).

A centrifuge is used to separate particles or macromolecules:

  • Cells
  • Sub-cellular components
  • Proteins
  • Nucleic acids

Basis of separation for a centrifuge are:

  • Size
  • Shape
  • Density

A centrifuge utilizes density difference between the particles/macromolecules and the medium in which these are dispersed and Dispersed systems are subjected to artificially induced gravitational fields.

 

TYPES OF CENTRIFUGATION

 

                                                Centrifugation

 

                      Preparative                                                               Analytical

 

                                 Differential              Density-gradient

                                                  

 

                                  Rate zonal                               Isopycni

 

Preparative Centrifugation; it is used to separate organelles and molecules and it can handle larger liquid volumes. It does not required any optical read-out.

Separation methods used in preparative ultracentrifugation are:

  • Differential Centrifugation; In this, separation is based on the size of the particles. This is commonly used in simple pelleting and in obtaining partially-pure preparation of subcellular organelles and macromolecules. For the subcellular organelles, tissue or cells are first disrupted to release their internal contents. This crude disrupted cell mixture is called as a homogenate. During centrifugation of a cell homogenate, larger particles sediment faster than smaller ones and this provides the basis for obtaining crude organelle fractions by differential centrifugation. When a cell homogenate is centrifuged at 1000 x g for 10 minutes, unbroken cells and heavy nuclei pellet to the bottom of the tube.

    The supernatant can be further centrifuged at 10,000 x g for 20 minutes to pellet subcellular organelles of intermediate velocities such as mitochondria, lysosomes, and microbodies. Some of these sedimenting organelles can obtained in partial purity and are typically contaminated with other particles. Repeated washing of the pellets by resuspending in isotonic solvents and re-pelleting may result in removal of contaminants that are smaller in size (Figure 1). Obtaining partially-purified organelles by differential centrifugation serves as the preliminary step for further purification using other types of centrifugal separation (density gradient separation).

Density gradient centrifugation; this method is preferred to purify subcellular organelles and macromolecules. Density gradients can be generated by placing layer after layer of gradient media such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top in either a discontinuous or continuous mode. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into two categories.

  • Rate-zonal (size) separation.
  • Isopycnic (density) separation.

Rate zonal (size) separation; also known as sucrose density gradient centrifugation,it uses particle size and mass instead of particle density for sedimentation. Figure 2 illustrates a rate-zonal separation process and the criteria for successful rate-zonal separation. Certain types of rotors are more applicable for this type of separation than others. Examples of common applications include

  • separation of cellular organelles such as endosomes or
  • separation of proteins, such as antibodies.

For instance, Antibody classes all have very similar densities, but different masses. Thus, separation based on mass will separate the different classes, whereas separation based on density will not be able to resolve these antibody classes.

Isopycnic separation; also known as caesium chloride density gradient centrigugation,in this type of separation, a particle of a particular density will sink during centrifugation until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. Once this quasi-equilibrium is reached, the length of centrifugation does not have any influence on the migration of the particle. A common example for this method is separation of nucleic acids in a CsCl gradient. Figure 3 illustrates the isopycnic separation and criteria for successful separation

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BOD Incubator- Determination of Biological Oxygen Demand

April 21st, 2014

BOD incubator is the most versatile and reliable low temperature Incubator which is designed to maintain at 20°C, necessary for Biological Oxygen Demand/Biochemical Oxygen Demand (BOD) determination. BOD incubators provide controlled temperature conditions for accelerated tests and exposures.  The biological oxygen demand (BOD) is an empirical test in which standardized laboratory procedures are used to determine the relative oxygen requirements of microbes in waste waters, effluents, and polluted waters and in simple words,.

It is a chemical process that determines how fast biological organisms use up oxygen in a body of water or it measures the oxygen required for the biochemical degradation of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic materials, such as sulfides and ferrous iron  The seeding and dilution procedures provide an estimate of the BOD at pH 6.5 to 7.5.

Overview of BOD Methodology

Biochemical oxygen demand (BOD), sometimes referred to as Biological Oxygen Demand, is a quantitative expression of microbes ability to deplete the oxygen content of a waste water. This depletion takes place due to the microbes consuming organic matter in the water via aerobic respiration. This type of respiration uses oxygen as an electron acceptor, and the organic material being consumed provides the energy source. This organic matter also undergoes oxidation without the aid of microbes, which can be measured using the Chemical Oxygen Demand (COD) procedure.

The method consists of placing a sample in a full, airtight bottle and incubating the bottle under specified conditions for a specific time. Dissolved Oxygen (DO) (amount of oxygen present in water body) is measured initially and after incubation. The BOD is computed from the difference between the initial and final DO.

High levels of BOD in streams cause the dissolved oxygen (DO) content of the water to drop. It is this DO that fish, and zooplankton use to survive. If DO drops to below a critical level the ecology of the stream could begin to die off as well. This condition can lead to an increase in anaerobic bacteria (species that can live in the absence of oxygen) that leads to the production of foul-smelling and possibly toxic gases. These gases may include methane, hydrogen sulfide, and ammonia. If this water were to enter into groundwater sources one can imagine the potential problems with toxicity to humans.

Factors affecting the BOD

Some factors may affect the accuracy and precision of BOD measurements. Such as

  • Soluble versus particulate organics,
  • Settle able and float able solids,
  • Oxidation of reduced iron and sulfur compounds, or
  • Lack of mixing
  • Oxidation of reduced forms of nitrogen, such as ammonia and organic nitrogen, can be mediated by microorganisms and exert nitrogenous demand. Nitrogenous demand historically has been considered as an interfering factor in the determination of BOD, and the inclusion of ammonia in the dilution water contributes an external source of nitrogenous demand. The interference from nitrogenous demand can now be prevented by an inhibitory chemical (If an inhibiting chemical is not used, the oxygen demand measured is the sum of carbonaceous and nitrogenous demands).

BOD and Water Pollution

Biological Oxygen Demand (BOD) is one of the most common measures of pollutant organic material in water. BOD indicates the amount of pollutants present in water. Therefore, a low BOD is an indicator of good quality water, while a high BOD indicates polluted water. Dissolved oxygen (DO) is consumed by bacteria when large amounts of organic matter from sewage or other discharges are present in the water. DO is the actual amount of oxygen available in dissolved form in the water. When the DO drops below a certain level, the life forms in that water are unable to continue at a normal rate. The decrease in the oxygen supply in the water has a negative effect on the aquatic life.

The BOD test serves an important function in stream pollution-control activities. It is a bioassay procedure that measures the amount of oxygen consumed by living organisms while they are utilizing the organic matter present in waste, under conditions similar in nature. The other traditional tests or indicators for water quality are Chemical Oxygen Demand (COD) and pH.

Applications

  • In measuring waste loadings to treatment plants and in evaluating the BOD-removal efficiency of such treatment systems.
  • It measures the molecular oxygen utilized during a specified incubation period for the biochemical degradation of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic material such as sulfides and ferrous iron.
  • It measures the amount of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless their oxidation is prevented by an inhibitor.

Safety Guidelines

  • Do not cover the shelves of the incubator with aluminum foil. It may cause temperature control malfunction or an obstruction of air circulation.
  • Control the temperature of incubator regularly to avoid any error.
  • While performing test BOD bottles should be closed in BOD incubator.
  • BOD incubators are not designed to store eating and drinking materials and cosmetics too.
  • Flammable substances are not permitted to be stored.

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