UV LIGHT EFFICIENCY TEST

             UV LIGHT EFFICIENCY TEST

1.0 METHODOLOGY

1.1  Prepare SCDA medium as per SOP for preparation of culture media.

1.2  Aseptically pour approximately 15-20 ml of sterile molten cooled (40 - 45°C) SCDA agar into sterile 90 mm petri plates under LAF.

1.3  Allow the media to solidify the plates under LAF, after solidification label all the plates with name of media, preparation batch No. and date of preparation.

1.4  Invert and incubate the plates at 30 to 35°C for 24 hrs in BOD Incubator. After incubation physically inspect the plates for any contamination (Macroscopic growth of evidence), if there is contamination discard the plates as per SOP for Destruction of Microbial waste by Autoclaving.

1.5  After pre-incubation, transfer 1 ml of not less than 105 cfu per ml culture of Bacillus subtilis to two SCDA Petri plates & spread the colony using sterile spreader. (Prepare 106 Bacillus subtilis cfu per ml suspension)

1.6  Now wrap one Petri plate containing Bacillus subtilis  in Aluminum foil and do not wrap other Petri plate. Now transfer both the Petri plates in LAF. Open the lid of unwrapped Petri plate.

1.7  Now switch on UV light for 30 minutes.

1.8  After 30 minutes exposure, switch off the UV light & close the lid of unwrapped Petri plate.

1.9  Incubate both the Petri plates in incubator at 30-35ºC for 24-48 hours.

1.10  Prepare positive control by streaking Bacillus subtilis culture and negative control as it is without streaking.

1.11  Apply the same procedure for UV Light of other LAF & Pass Box. For checking Efficiency of UV Light of LAF of Sterility Room, do not perform the test in Sterility Room. For the test, the UV light of Sterility LAF should be fitted to MLT or Bioassay LAF.

1.12  After incubation observe the plates for total bacterial count. Record the results.

2.0  PRECAUTIONS

2.1  Switch on the UV light after exposing the plates on the work station of the LAF.
2.2  Switch off the UV light before collecting the plates.

3.0 FREQUENCY
3.1 Once in a month.

REMOVAL OF RESIDUAL CHLORINE BEFORE MICROBIAL ANALYSIS

REMOVAL OF RESIDUAL CHLORINE BEFORE MICROBIAL ANALYSIS

It is important to remove the residual chlorine from the water sample before the microbial analysis.
Raw water is chlorinated by adding sodium hypochlorite (NaOCl) to make its concentration 2-4 ppm. Chlorination of water is done to remove the microbial load from the water. It helps the water system to produce the purified water of specified microbial quality.

MECHANISM OF ACTION:-

When sodium hypochlorite is dissolved in water, it produces hypochlorous acid and hypochlorite ion. These hypochlorite ions are known as “free chlorine”. This free chlorine is responsible for the chlorination of the microbes in water. These ions are highly active and penetrate through the cell wall via lipids and destroy the enzymes in the cells.

All the free chlorine is not consumed in the chlorination. This remaining chlorine is known as "residual chlorine" which can interfere with the microbial analysis of the water. This may alter the actual results.

Sodium thiosulfate (Na2S2O3) can be used to neutralize or remove residual chlorine. It simply reacts and forms sodium hydrogen sulfate or sodium bisulfate which is an inactive salt.

Na2S2O3 + 4HOCl + H2O -----> 2NaHSO4 + 4HCl

Add 0.1 ml of 10% solution of sodium thiosulfate is added to the water sample bottle before sterilization. This can be prepared by dissolving 100 gm of sodium thiosulfate crystals in about 500 ml of water making up a final volume to 1000 ml. This solution can be stored under refrigeration for six months.

This neutralization of residual chlorine will help to determine the actual microbial load in water sample and will not affect the microbial recovery.

ACTIVATED CARBON FILTER

Activated Carbon Filter and Increase in Efficiency for Water System

Activated carbon filters are used for water purification in water systems and application of these filters can increase the efficiency of water system.

Activated carbon filter is used to remove chlorine and organic matter from the water. It also removes the color and odor from the water hence the turbidity of water is also removed. It plays an important role in water purification systems where chlorine is added for decontamination. ACF is unable to remove the microbes and hardness.
Activated carbon filter contains carbon particles of various sizes ranging from 10 to 50 mesh. Activated carbon is prepared from coconut shells by burning in the presence of oxygen. This carbon is then activated by steam or chemical methods. In steam activation this carbon is heated at 750- 950°C in the presence of steam. Steam activated carbon contains very fine pore size that is ideal for adsorption of components from water.

In chemical activation of carbon, it is mixed with a paste of zinc chloride and heated at 600-800°C. Chemically activated carbon has bigger pore size and ideal for adsorption of larger molecules from water.

Chlorine and organic compounds are adsorbed on the surface of activated carbon particles into their small pores. Reactivation of the carbon required again to increase the efficiency of activated carbon filter frequently.

There are some factors that affect the efficiency of the carbon filters such as contact time of water on carbon bed, concentration of contamination, temperature and pH of water. At high temperature activated carbon filter is less effective then low temperature. pH of water also affect the adsorption of contaminates. A 20 % more activated carbon surface is required when we increase pH by one unit from 7.

Sanitation of activated carbon filters should be done frequently with the steam because carbon supports the growth of microbes and it can contaminate the water systems.

BUBBLE POINT TEST

                       BUBBLE POINT TEST

Membrane filters have been used successfully for many years to remove yeast, bacteria and particulate from fluid streams. The ultimate integrity test for a sterilizing grade membrane filter is the bacterial challenge. Unfortunately this is a destructive test; the filter cannot be used afterwards to filter a product. As a result, one of the most important aspects of the use of filters to remove bacteria is to have a non-destructive integrity test, which is correlated to the production of effluent in a bacterial challenge test. It is this correlation, rather than the nondestructive integrity test alone, that provides assurance that the filter will perform as intended. During production of sterile product, the filter should be subjected to such an integrity test before and after filtration. This is done to ensure that the filter meets specification, is properly installed and intact during filtration, and to confirm the rating of the filter. There are 3 major tests used to determine the integrity of a membrane filter: the Bubble Point Test, the Forward Flow Test, and the Pressure Hold Test. All three tests are based on the same physics, the flow of a gas through a liquid-wetted membrane under applied gas pressures. They differ in which part of the flow/pressure spectrum they examine. In this paper, we will focus on the Bubble Point Test. Background A bubble point test is a test designed to determine the pressure at which a continuous stream of bubbles is initially seen downstream of a wetted filter under gas pressure. To perform a Bubble Point Test, gas is applied to one side of a wetted filter, with the tubing downstream of the filter submerged in a bucket of water. The filter must be wetted uniformly such that water fills all the voids within the filter media. When gas pressure is applied to one side of the membrane, the test gas will dissolve into the water, to an extent determined by the solubility of the gas in water. Downstream of the filter, the pressure is lower. Therefore the gas in the water on the downstream side is driven out of solution. As the applied upstream gas pressure is increased, the diffusive flow downstream increases proportionally. At some point, the pressure becomes great enough to expel the water from one or more passageways establishing a path for the bulk flow of air. As a result, a steady stream of bubbles should be seen exiting the submerged tubing. The pressure at which this steady stream is noticed is referred to as the bubble point. Bubble Point Test Procedure A forward bubble point integrity test is a procedure which measures the pressure needed to be applied to the upstream side of a filter causing bulk or open pore flow through the largest pores of a wetted filter. This measurement is taken from the downstream side of the filter housing where a flexible piece of tubing has been attached and the other end submerged into a beaker of water. The bubble point is indicated by vigorous bubbling from the tubing. The accuracy of this test will rely on the operator’s ability to successful recognize this point.

Test Method

1. Record the filter part number(s), lot number, and product information. Also include physical observations.

2. Wet the filter to be tested with water.

3. Place the wetted filter in the appropriate housing.

 4. Connect the outlet fitting from the compressed air pressure regulator to the upstream side of the test filter. Check that the gauge which is connected to the pressure regulator has subdivisions of at least 0.5 psig, and has the capacity to measure up to 100 psig. A digital pressure gauge can also be used.

 5. Connect the outlet fitting from the compressed air pressure regulator to the upstream side of the test filter.

6. Connect a piece of flexible tubing from the downstream port of the test filter into a beaker filled with water.

7. Starting from zero pressure, gradually increase the pressure to the test filter using the pressure regulator.

8. Observe the submerged end of the tubing for the production of bubbles as the upstream pressure is slowly increased in 0.5 psig increments. Note the rate that the bubbles appear for the end of the submerged tube.

9. The bubble point of the test filter is reached when bubbles are produced from the tube at a steady rate. Record the pressure to the nearest 0.5 psig as indicated on the pressure gauge. If the recorded pressure is greater than or equal to the minimum expected bubble point listed above, the filter is integral. If the recorded pressure is lower than the minimum bubble point listed above, the filter has failed the integrity test.