WHY PETRI DISHES INVERTED DURING INCUBATION

Petri dishes having media are incubated in inverted position after inoculation. This has different advantages those improve the result accuracy.

Petri dishes were first used by German physician Julius Petri in 1887. He covered one culture plate to another plate to prevent the contamination. Before him the culture plates were covered with the glass bell jars.

There are following advantages during incubation when we incubate the plates in inverted position.

1. When the plates are incubated in normal position, water evaporation from media occurs. These vapors condense on the lid of the Petri dish and drops fall on the colonies developed on the media surface. This causes colony mix-up with each other and spread throughout the plate surface. This creates problem in counting and proper determination of microbial count.

2. The evaporation of water from media can cause media dryness that can affect the microbial growth but when the plates are incubated in inverted position, the rate of evaporation decreases that results in proper microbial growth. Therefore, Petri dishes with media can also be stored for a longer period in inverted position.

3. The lid of Petri dishes may contain any contamination that spreads on the media and grows with the sample microbes. This may create an error in microbial count determination.

4. It is easy to handle the inverted Petri dishes because the lid of the Petri dish may open during handling when incubated in normal position and it may cause contamination from air. We label the Petri dishes at the bottom part because lid may exchange with other Petri dishes creating confusion and inverted position makes it easy to read the labeling of Petriplates.

PHARMACEUTICAL WATER

INTRODUCTION

Water is the one of the major commodities used by the pharmaceutical industry. It is widely used as a raw material, ingredient, and solvent in the processing, formulation, and manufacture of pharmaceutical products, active pharmaceutical ingredients (APIs) and intermediates, and analytical reagents. It may present as an excipient, or used for reconstitution of products, during synthesis, during production of finished product, or as a cleaning agent for rinsing vessels, equipment and primary packing materials etc. There are many different grades of water used for pharmaceutical purposes. Several are described in USP monographs that specify uses, acceptable methods of preparation, and quality attributes. These waters can be divided into two general types: bulk waters, which are typically produced on site where they are used; and packaged waters, which are produced, packaged, and sterilized to preserve microbial quality throughout their packaged shelf life. There are several specialized types of packaged waters, differing in their designated applications, packaging limitations, and other quality attributes. Different grades of water quality are required depending on the different pharmaceutical uses.
There are also other types of water for which there are no monographs. These are all bulk waters, with names given for descriptive purposes only. Many of these waters are used in specific analytical methods. These nonmonographed waters may not necessarily adhere strictly to the stated or implied modes of preparation or attributes.

TYPES OF WATER USED:-

Water is the most common aqueous vehicle used in pharmaceuticals. There are several types of water are used in the preparation of drug product, such as;

NON-POTABLE WATER

Non-potable water is water that is not of drinking water quality, but which may still be used for many other purposes, depending on its quality. Non-potable water is generally all raw water that is untreated, such as that from lakes, rivers, ground water, springs and ground wells.

Purposes:-
•  cleaning of outer surface of the factory
•  used in garden
•  washing vehicles etc

POTABLE WATER

Portable water is not suitable for general pharmaceutical use because of the considerable amount of dissolved solids present. These dissolved solids consist chiefly of the chlorides, sulphates and bicarbonates of Na, K, Ca and Mg. A 100 ml portion of official water contains not more than 100 mg of residue (0.1%) after evaporation to dryness on a steam bath.

Purposes:-
•  To use as drinking water
•  Washing and the extraction of crude drugs
•  Preparation of products for external use

PURIFIED WATER

Purified water is used in the preparation of all medication containing water except ampoules, injections, some official external preparations such as liniments. Purified Water must meet the requirements for ionic and organic chemical purity and must be protected from microbial contamination. The minimal quality of source or feed water for the production of Purified Water is Drinking Water.

Purposes:-

•  For the Production of non-parenteral  preparation/formulation
•  For the Cleaning of certain equipment used in non-parenteral product preparation
•  For Cleaning of non-parenteral product-contact components
•  For All types of tests & assay
•  For the Preparation of some bulk chemicals
•  For the preparation of media in microbiology laboratories

Preparation technique:-

•  Deionization
•  Distillation
•  Ion exchange

WATER FOR INJECTION(WFI)

Water for Injection is a solvent used in the production of parenteral and other preparations where product endotoxin content must be controlled, and in other pharmaceutical applications Water For Injection (WFI) is sterile, non pyrogenic, distilled water for the preparation of products for parenteral use. It contains no added substance and meets all the requirements of the tests for purified water. It must meet the requirements of the pyrogen test. The finished water must meet all of the chemical requirements for Purified Water as well as an additional bacterial endotoxin specification. Since endotoxins are produced by the kinds of microorganisms that are prone to inhabit water, the equipment and procedures used by the system to purify, store, and distribute Water for Injection must be designed to minimize or prevent microbial contamination as well as remove incoming endotoxins from the starting water. Water for Injection systems must be validated to reliably and consistently produce and distribute this quality of water.

Purposes:-

(i) For the production of parenteral products/formulation
(ii) For cleaning of parenteral product-contact components.

Preparation technique:-
• Distillation
• Reverse osmosis
• Membrane process

Storage condition

It can be stored for periods up to a month in special tanks containing ultraviolet lamps. When this freshly prepared water is stored and sterilized in hermitically sealed containers, it will remain in good condition indefinitely.
If autoclave is not available, freshly distilled water may be sterilized by boiling the water for at least 60 minutes in a flask stoppered with a plug of purified non absorbent cotton covered with gauze, tin-foil or stout non absorbent paper; or the neck of the flask may be covered with cellophane and tightly fastened with cord.

STERILE WATER FOR INJECTION

It’s specifications are provided in USP monograph for water for injection, sterilized and packaged in suitable single-dose containers, preferably of type I glass, of not larger than 1000 ml size. It meets the requirements of the sterility test and pyrogen test and other tests under purified water.

Purposes:-

• Used for extemporaneous preparation compounding.
• Used as a sterile diluents for parenteral products.

Preparation technique:-

• By distillation of water for injection (WFI)

BACTERIOSTATIC WFI

This is sterile Water for Injection containing bacteriostatic (antimicrobial) agents. It may be packed in single-dose containers of not larger than 5 ml size and in multiple-dose containers of not larger than 30 ml size, the label of which indicates the name and the proportion of added agent.

Purposes:-

• Used as a diluents in the preparation of parenteral products

Preparation technique:-

• By using sterile water for injection

STERILE WATER FOR INHALATION

Sterile water for Inhalation is Water for Injection that is packaged and rendered sterile and is intended for use in inhalators and in the preparation of inhalation solutions. It carries a less stringent specification for bacterial endotoxins than Sterile Water for Injection, and therefore, is not suitable for parenteral applications.

STERILE WATER FOR IRRIGATION

Sterile water for irrigations is Water for Injection packaged and sterilized in single-dose containers of larger than 1 L in size that allows rapid delivery of its contents. It need not meet the requirement under small volume injection.

Purposes:-

• To bath and moisten body tissue.
• Performing urologic procedure for surgeon.

Preparation technique:-

• From water for injection

Example:-

• Surgical irrigation solution (Splash solution)
• Urologic irrigation solution
• Glycine solution
• Sorbitol solution

WATER FOR HAEMODIALYSIS

Water for hemodialysis is used for hemodialysis applications. It may be packaged and stored in unreactive containers that preclude bacterial entry. The term “unreactive containers” implies that the container, especially its water contact surfaces, are not changed in any way by the water, such as by leaching of container-related compounds into the water or by any chemical reaction or corrosion caused by the water. The water contains no added antimicrobials and is not intended for injection.

Purposes:-

• For the dilution of hemodialysis concentrate solution.

Preparation technique:-

• From safe drinking water.

PURE STEAM
 
Pure steam is also sometimes referred to as “clean steam”.

Purposes:

• To remove any co-deposited impurity residues.
• For air humidification in controlled manufacturing environments.
• Used in steam sterilization of equipment and porous loads.
• For cleaning the places where condensate directly comes in contact with official articles, product contact containers, and surfaces.

WATER MISCIBLE SOLVENTS

Although water miscible solvents are used in parenterals, principally to enhance drug solubility, it is important to mention that they also serve as stabilizers for those drugs that degrade by hydrolysis. A water miscible solvent must be selected with grade care for it must not be irritating, toxic, or sensitizing, and it must not exert an adverse effect on the ingredients of the formulation.

• Solvents that are miscible with water are:

         dioxolanes
         dimethylacetamide
         butylene glycol
         polyethylene glycol 400 and 600
         propylene glycol
         glycerin and
         ethyl alcohol

• Water immiscible solvents include:
        Fixed oil
        ethyl oleate
        Isopropyl myristate, and
        Benzyl benzoate.

WHY 70% IPA USED IN PHARMA

70% isopropyl alcohol is most commonly used disinfectant in pharmaceutical industries. The important thing is that only 70% solution of isopropyl alcohol acts as a disinfectant killing all surface microorganisms. It is used to disinfect hands and equipment surface in pharma.

70 % isopropyl alcohol solution kills microorganisms by dissolving plasma membrane of the cell wall. Plasma membrane of gram negative bacteria consist of thin layer of peptidoglycon that easily destroyed by the alcohol. Therefore, 70 percent iso propyl alcohol is known as pharmaceutical alcohol.

Water is also required to denature the proteins of cell membrane and acts as a catalyst in the reaction. Contact time of the alcohol with the organism also play an important role. A 70% solution of alcohol takes more time in evaporation from the surface, increasing the contact time. Therefore, 70% isopropyl alcohol fulfills the both requirements.

100% isopropyl alcohol coagulates the protein instantly creating a protein layer that protects the remaining protein from further coagulation. Due to this organism is not killed but remains in dormant stage. While 70% isopropyl alcohol solution penetrates in the cell wall at slower rate and coagulates the all protein of the cell wall and microorganism dies.

Thus 70% IPA solution in water is more effective then 100% absolute alcohol and have more disinfectant capacity.

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.