STAINING TECHNIQUES IN MICROBIOLOGY

STAINING TECHNIQUES IN MICROBIOLOGY

STAINS (DYES)

·         Stains are organic compounds carrying either positive charges or negative charges or both.

·         Based on the charge they have stains are categorized as follows

Basic Dyes : stains with positive charge (Methylene Blue, Crystal violet) – Background staining.

Acidic Dyes : stains with negative charge (India ink, Nigrosine)- Bacterial cell staining.

Neutral Dyes : stains with both charges (Eosinate of methylene blue, Giesma sain)- Stains nucleic acid and cytoplasm.

·         The word DYE is used to describe the crude form of colouring agent used for general purpose and the word STAIN used to describe the pure form of colouring agent commonly used for biological staining purpose.

·         In a normal natural smear bacteria are not clearly visible under microscope because they have nearly the same refractive index as water as the bacterial cytoplasm is transparent. Therefore we are applying different types of stains for make them clearly visible under microscope.

·         Every stains used for staining have two common properties

                                      I.        Stains have a chromophore group which give the dye its colour.

                                    II.        They can bind with bacterial cell wall through ionic, covalent or hydrophobic bonds.

PURPOSE OF STAINING MICROBIAL CELL

·         To understand the size and shape of a bacterial cell.

·         To enhance the visualization of bacterial cells or specific structural component of a cell.

·         To highlight metabolic process of a cell.

·         Dead and live cell differentiation from a given sample.

·         Detailed study of internal or external structures of a cell.

·         To differentiate different types of cells from a single source.

MAJOR STAINING TECHNIQUES

·         Staining is simply explained as coloured chemical compounds used for making a contrast between the specimen and the background.

·         Microbiologists developed different types of staining techniques in microbiology for the clear visualization of different microbes.

·         It give specific colour to bacteria or its structures and make it clearly visible under microscope – simply bacterial staining means colouring of bacteria to make it clearly visible under microscope.

·         Basic Requirements for Staining

i)      Clean and grease free glass slide

ii)     Bacterial culture to be stained.

iii)    Inoculation loop for the transfer of culture from suspension to slide.

iv)   Bunsen burner for sterilizing the inoculation loop and for heat fixation.

 DIFFERENT TYPES OF STAINING AND THEIR PURPOSE

Sr. No.	Types Of Staining	Staining Purpose	Chemicals used/ Example
1	Simple Staining	To understand the size, shape and cell arrangements. To understand the total count of bacterial cell in a given specimen.	Methylene blue Crystal violet Carbol fuchsin
2	Gram Staining	To differentiate between gram positive (purple colour) and gram negative (pink colour) bacterial cells from a given sample.	Crystal violet Safranin
3	Acid fast Staining	To identify the bacteria which have huge amount of lipids in their cellwall.	Used for the staining of Mycobacterium tuberculosis    and Mycobacterium leprae
4	Special staining	For the identification of special structural features of bacterial cells.	Endospore staining Capsule staining Flagella staining.
5	Iron-Heamatoxylin Staining	Staining of tissue components.	Staining of myelin, elastic and collagenic fibres etc.
6	Wheatley Trachoma Staining	Easiest method of staining protozoa	Detection and identification of intestinal protozoa.
7	Positive staining of Viruses	To observe and identify viruses under electron microscope.	Analysing morphological characters of virus from an aquatic sample.
8	Negative Staining Of Viruses	To observe and identify viruses under electron microscope.	Used to obtain high quality micrographs.
9	Gomori  Methenamine  Silver (GMS) Staining	To observe and identify fungi from tissue and incytology specimens.	Microscopic identification of Aspergillus.
10	Periodic acid-Schiff (PAS )  Staining	To observe and identify fungi from tissue and incytology specimens.	Cryptococcus staining.

ASEPTIC PROCESS SIMULATION/ MEDIA FILL

ASEPTIC PROCESS

Aseptic process is the manufacturing of sterile drug product using aseptic techniques with the sterile product coming in contact with only sterile components and exposed to only in sterile environment.

ASEPTIC PROCESS SIMULATION

Aseptic process simulation is simulation /mimics the aseptic processing as closely as possible, while sterile media is using instead of drug product.

PURPOSE OF ASEPTIC PROCESS SIMULATION

•       A media fill run is done to validate the aseptic techniques and aseptic process which is to be carried out during routine production.

•       To evaluate the aseptic process is capable to produce the sterile product.

•       To qualify the operators and asses their aseptic techniques.

MEDIA USED FOR ASEPTIC PROCESS SIMULATION

Generally soybean casein digest medium is used for media fill, because SCDM is a good growth medium for common bacteria and fungi. Any possible contaminant in the system or introduced during the aseptic processing is caught up in the media and multiplied, thereafter identified by naked eye  too.  

Why SCDM?

•       Wide range of microorganism growth

•       GPT

•       Solubility

•       Clarity

•       Concentration

•       Filterability

•       Availability

MEDIA FILL FREQUENCY

•       Three consecutive separate successful run during initial qualification, thereafter routine semiannual qualification for each line.

•       Any change to line or process which can affect the aseptic process should be validated through additional media fills, which include

                      I.        Facility modification

                    II.        Equipment modification

                   III.        Line configuration change

                  IV.        OOS environmental monitoring

                    V.        Extended shut downs

                  VI.        Sterility test failure

SIZE OF MEDIA FILL

A generally acceptable starting point for run size is in range of 5,000 to10, 000 units.

•       If the commercial batch size is under 5,000 then the media fill units should be equal to lager batch size.

•       If the commercial batch size is > 5,000 to <10,000 then the media fill units should be NLT 5,000.

•       If the commercial batch size is more than 10,000 then the media fill units should be NLT 10,000 units.

Sandwiching theory:-

Agalloco claims that the USFDA Suggested sandwiching WFI filling in between media fill.

 Example – If the largest commercial batch size is 100000 then media fill can be done by 5000 units with media 90000 units with WFI and end with 5000 units with media.

MEDIA FILL DURATION

As per ISO

“The process simulation run shall be of sufficient duration to cover the most routine manipulations and operation as well as the greatest possible number of permitted interventions”.

As per PDA

“Media fill duration should be of sufficient duration to fill sufficient number of units for proper contamination rate determination”.

LINE SPEED DURING MEDIA FILL

There is no specification available for line speed, but USFDA suggested high speed for processes with high manual interventions and slow speed for product with prolonged exposure of sterile drug.

INTERVENTIONS

•       All the interventions which can harm the sterility of the system /product during the production run are to be simulated in the media fill.

•       Any unplanned intervention should also be recorded

•       The respective units /vials to be labeled accordingly.

INCUBATION OF FILLED UNITS AND ITS EXAMINATION

•       Incubates the media units at 20-25 °c for 7 days and further at 30-35 °c for again 7days.

•       The vials are inverted every third day for maximum exposure of media to inside of vial.

•       All the vials are observed for growth visually by a trained microbiologist, both at the end of incubation at 20-25 °c and after incubation at 30-35 °c

INTERPRETATION OF RESULTS

•       When filling less than 5000 units, no contaminated units should be detected, one contaminated unit is considered cause an investigation following by revalidation.

•       When filling from 5000 to 10000 units,

1. One contaminated unit should result in an investigation including consideration of repeat media fill.

2. Two contaminated units are considered cause for revalidation following an investigation

•       When filling more than 10,000 units,

                          1. One contaminated unit should result in an investigation.

2. Two contaminated units are considered cause for revalidation following an       investigation.

MEDIA FILL FAILURE

•       The filling line is to be shut down immediately.

•       All the bathes filled since the media fill should be kept on hold.

•       The contaminant should be identified till species level.

•       A thorough investigation should be  done to ascertain the source of the contamination as per the protocol for the media fill failure investigation

•       Five production batches filled prior to the media fill should also be kept on hold.

•       All the held batches are subjected to extensive testing as per protocol.

•       Upon identifying the source of contamination, further three successful media fill to be taken.  

SEWAGE TREATMENT

SEWAGE

Water from community containing liquid and solid excreta derived from houses, streets washings, factories and industries.

-          Sullage : waste water does not contain human excreta.

-          Sewage or waste water includes the wastes such as

                                       i.        Human          : faecal material, urine

                                      ii.        Domestic      : food wastes, wash water

                                    iii.        Farm              :  fertilizers, pesticides

                                    iv.        Industrial      : acids, oils, greases.

-          Waste water containing water from sinks, tub, dish washers, showers, cloth washers etc are known as grey water.

-          Waste water containing water used for flush toilets, water containing human wastes are known as black water.

-          Composition of sewage varies depends on source that affects variation in microflora.

-          Almost all groups of microbes are present in sewage such as;

                                       i.        Algae

                                      ii.        Fungi

                                    iii.        Protozoa

                                    iv.        Bacteria &

                                      v.        Viruses.

   A.   COMPOSITION OF SEWAGE

1.    Chemical composition

o   Water (99.9%)

o   Solids (0.1%)

·         Organic solids (70%) – either nitrogenous (protein, aminoacids) or non nitrogenous (lipids, carbohydrates).

·         Inorganic solids (30%) – ammonia, chloride salts, metals etc.

2.    Microbial composition

o   Composition of microorganisms vary according to source of water.

o   Normally comprises bacteria, fungi, protozoa, algae, and viruses.

Microorganisms present in sewage
Algae	Fungus	Bacteria	Protozoa	Virus
Chlorella sp. Spirulina sp. Scenedesmus sp. Euglena sp. Oscillatoria sp.	Zoophagus sp. Geotrichum sp. Candida sp. Penicillium sp. Alternaria sp.	Nitrosomonas sp. Achromobacter sp. Pseudomonas sp. Nocardia sp. Leptothrix sp. Spaerotilus sp.	Vorticella microstoma Acineria uncinata Amphileptus pleurosigma Opercularia microdiscum Aspidisca cicada Paramecium caudatum	Enterovirus Hepatovirus Adenovirus Torovirus Reovirus Calicivirus

Testing of sewage for :       *        Total solids                   *        Nitrogen       *        Chlorides                 *        Dissolved oxygen       *        Organic carbon                     *        Biological oxygen demand       *        Chemical oxygen demand       *        Hydrogen sulphides           *        Grease & fats

Sewage treatment involves :      1.    Primary/Preliminary treatment – Physical removal of solids      2.    Secondary treatment – Biological decomposition of organic compounds      3.    Tertiary treatment – removal of heavy metals, nitrates, phosphates etc.

   B.   SMALL SCALE SEWAGE TREATMENT

          1)    Cesspools 

•          Household treatment.
•          Wastes are directly thrown into cesspools in many homes.
•      It is constructed in underground part with concrete and contains wall of cylindrical rings with pores.
•          Waste water or sewage enters the cesspool through the inlet pipe.
•          Bottom remains open.
•         Suspended solid materials fall on the bottom of cesspool subsequently and forms sludge after getting deposited in huge amount.
•          Water passes out into surrounding soil through open bottom and pores of cesspool.
•       Anaerobic bacteria decomposes the organic materials of sludge resulting in the       release and deposition of breakdown products on the ground.
•        When the amount of breakdown products exceeds and forms a thick layer, the bottom need to be cleaned by strong acids.
•       Dried microbial preparations (eg; Bacillus subtilis, yeast cells.) can be added at intervals for accelerating the decomposition of sludge deposited at the bottom of cesspool.

2)    Septic tank

•           A water tight tank into which household sewage is admitted for treatment.
•          These settling tanks are based on sedimentation principle.
•           The tanks may be metallic or concrete constructed below the ground level.
•           One of the earlist process developed for treating waste water.
•           The domestic wastes flow through the inlet pipes into septic tanks.
•           The suspended organic materials are accumulated at the bottom of tank whereas the water flows out through outlet to a distribution box.
•          Distribution box is connected with perforated pipes that open under the soil surface in the surrounding  areas.
•           Drinking water must be kept at certain distance of the pipes of septic tank as pathogenic microbes are not eliminated.
•          Anaerobic decomposition occurs in which complex organic matters are converted into simple molecules and also into gases.
•          Two processes; sedimentation and biodegradation (anaerobic digestion & aerobic oxidation) are accomplished in septic tank.

Sedimentation :

*        The solid material present in the waste water suspended at the bottom of the tank.

*        These suspended materials undergo biodegradation.

Anaerobic digestion :

*        This is the first stage of purification that occurs inside the septic tank.

*        Human excreta consist of 65% mineral matter (do not undergo any chemical change in a septic tank) and 35% organic matter (of which 20-40% of organic solids are liquefied or gasified in the septic tank.

*        Heavier matter settles at the bottom called sludge and lighter matter like grease, fats etc. form a layer at the top called scum.

*        Solids or sludge are attacked by anaerobic microbes and broken down into simpler forms.

*        After anaerobic digestion gases are formed (mostly methane) which rises to the surface as bubbles.

Aerobic oxidation :

*        Occurs outside the septic tank (ie., in subsoil).

*        The liquid which passes out of the outlet pipe from time to time called effluent.

*        It contains numerous bacteria, cysts, helminthes, and organic matters.

*        The effluent is allowed to percolate in the subsoil.

*        There are millions of bacteria in layers of soil which attack the organic matter present in the effluent.

*        Thus organic matter is oxidized into stable end products like nitrates, CO₂, H₂O etc.

3)    Imhoff’s tank

•           Improved design of septic tank that maintains anaerobic condition.
•           Produces some utilizable bio gas and facilitates the settling of solids.
•           The tank has three compartments ;

*        Upper compartment or settling compartment and

*        Lower compartment or digestion compartment.

*        The gas vent and scum compartment.

•           Upper and lower compartments are separated from each other.
•           When sewage is introduced in the upper compartment that settles down and slips by an opening into the lower compartment and digested.
•           Digested solids are removed from the bottom of the tank periodically.
•           It provide a suspended solid waste removal upto 60 percentage and BOD removal upto 35 percentage.
•           Usually Imhoff’s tanks are 9 meter deep, construction is costly and expert maintenance is required.

C.   LARGE SCALE SEWAGE TREATMENT

1.    Primary / Preliminary treatment

•           Physical removal of solids (organic and inorganic).
•           Approximately 40 to 60 percent suspended solid materials are removed from the influent waste water.
•           Consist of screening and sedimentation.

a.    Screening

•          Remove larger floating solids and organic solids which do not aerate and decompose and to skim grease and fatty acids.
•          Types of screeners used :

*        Rock or coarse screens (75mm).

*        Medium screens (12.5 – 40mm).

*        Fine screens (13mm).

•           Solids are removed at regular intervals and the screen is slowly rotated to prevent overloading.
•           Disposal of screenings :

*        Dumping into sea.

*        Incineration.

*        Burial to shallow trenches.

*        By mixing the screenings with house refuse composting.

b.    Grit chamber

•           Removal of grit based on the principle of differential settling.
•           It removes heavy inorganic matters like grit, sand, gravel, road scrapings and ashes.
•          These particles may injure pipes and make sludge digestion difficult.
•          Grit particles are of high density when compared to organic matter and settle at a velocity of 1.2mm/min.
•           Length of tank – 18m.
•           Clearing period – 2 weeks.

c.    Skimming tanks

•           Sewage contains lot of grease and fatty acids that form a layer at the top of the tank called scum that interfere with oxidation processes.
•           These scum layers removed by treating in skimming tank or primary settling basins.
•           Tanks may built in 1m depth.
•           Scum accumulations are removed manually or mechanically which then buried or burnt.

d.    Sedimentation

•           It is a very large rectangular tank works on the principle of settle down under gravity.
•           Nearly 50 –70% of solids are removed.
•           A reduction between 30 – 40% in the number of coli forms is obtained.
•           The organic matter which settles down called sludge is removed by mechanical devices without disturbing operation of tank.
•           Removal of suspended solids reduce the strength of the sewage.
•           Settling occurs when water stands still or flows slowly through a basin or tank.
•           High density particles settle down under gravity and form sludge at the bottom of tank whereas clarified water will be collected through the outlet.
•           Types of tanks :

*        Vertical flow tank

*        Horizontal flow tank

*        Circular or radial flow tank

2.    Secondary treatment

•           Biological decomposition of organic compounds.
•           Effluent from primary treatment tank still contains a proportion of organic matter as solution or colloidal state and numerous living organisms.
•           This treatment stage removes remaining solids and nutrients though biological decomposition.
•           Before being discharged into any water bodies it is necessary to oxidize the organic matter.
•           This oxidation is either carried out on land (naturally) or in bacterial beds (artificially).
•           Secondary treatment relies on microbial activity.
•           Since these oxidation processes are carried out by microorganisms they are called biological oxidators.

a.    Fixed film sewage treatment

                              i.        Biological filters / trickling filters

•           Also called sprinkling filter or percolating filter or bacteria bed.
•           Works on the principle of filtration over porous material.
•           Sewage is distributed by a sprinkler revolving over a bed of porous materials.
•           It is an artificially constructed bed consisting of broken stones, bricks or other suitable materials.
•           The organic matter oxidizes and nitrifies by the aerobic life that flourishes on the bio film.
•           During the flow of sewage over the surface of bed the aerobic bacteria lying dormant in the liquid become active and start breeding readily in favorable conditions and leads to the formation of a film called zoogleal film on the surface of bed.
•           Sewage percolate the porous bed and the effluent is collected at the bottom which finally results in growth of dense slimy bacteria that coats the porous material.
•           Example :Zooglea ramigera – has a principle role in generating slime matrix through secretion of exopolysaccharide which accumulates a heterogeneous microbial community.
•           This microbial community or slime layer absorbs and mineralizes the dissolved organic nutrients in the sewage thus reduces the biological oxygen demand of the effluent.
•          Effectively remove BOD upto 90 percentage.
•          A food web is established based upon the microbial film.
•           Sewage can be recirculated several times through the same filter for further clearance.

Drawbacks *        Nutrient overload may lead to excess microbial slime that reduces aeration and percolation rate. *        It can not be used during cold atmospheric condition. When the temperature is very low growth rate of microorganisms also become low.

Microorganisms commonly involved *        Pseudomonas sp. *        Flavobacterium sp. *        Achromobacter sp. *        Fusarium sp. *        Penicillium sp. *        Geotricum sp. *        Sporotricum sp. *        Chlorella sp. *        Ulothrix sp.

                            ii.        Rotating biological contractor / biodisc system

•           Closely spaced discs manufactured from plastic materials or other suitable material are rotated in a trough containing sewage effluent.
•           These discs are only partially submerged in effluent and they become coated with a microbial slime.
•           Continuous rotation of discs maintains the slime well aerated and contact with the sewage as a result bio films develop on the rotating biological contractors.
•          The bio film has two layers :

*        Outer aerobic whitish layer (eg; Beggiatoa sp.)

*        Inner anaerobic blackish layer (eg; Desulfovibrio sp.)

•           The blackish colour is due to the precipitation of iron sulphide, when iron reacts with hydrogen sulphide produced by sulphate reducing microorganisms, eg; Desulfovibrio sp.
•           The organic acids and alcohols, produced by fermentative microbes of anaerobic zone, are used up by sulphate reducing microbes and produces hydrogen sulphide which in turn diffuses into the outer aerobic zone where it is utilized by aerobic zone microbes.
•           Thickness of slime layer in all film flow process is governed by the diffusion of nutrients through the film.

-          Microorganisms involved :

*        Sphaerotilus sp.

*        Beggiatoa sp.

*        Nocardia sp.

*        Oscillatoria sp.

*        Desulphovibrio sp.

b.    Suspended cell sewage treatment

          Oxidation pond

•           Also known as oxidation lagoons or stabilization ponds.
•           Used for simple secondary treatment of sewage effluent.
•           In an oxidation pond, heterotrophic bacteria degrade organic matter in the sewage result in the production of cellular material and minerals which support the growth of algae.
•           These algal population allows further decomposition of organic matter by producing oxygen.
•           The production of this oxygen replenishes the oxygen used by the heterotrophic bacteria.
•           In order to support the algal growth the ponds need to be less than 10 feet deep.
•           Oxidation ponds are influenced by seasonal temperature changes therefore these are restricted to warmer climatic regions.
•           In this system, microbes grow as suspended particles within the water column rather than a biofilm.
•           Sewage is subjected to primary settling and is subsequently channelled through a series of oxidation pond.

•           Algae growing are :

*        Chlorella sp.

*        Spirulina sp.

*        Scenedesmus sp.

•          Based on activity oxidation ponds are :

*        Facultative ponds

*        Maturation ponds

*        Anaerobic ponds

*        Aerated ponds.

c.    Activated sludge process

•           Widely used aerobic treatment system
•           Sewage is passed into an aeration tank from primary settling tank which is then aerated by mechanical stirrer which causes floc formation.
•           The colloidal and finely suspended matter of sewage from aggregates are called floccules are permitted to settle down in secondary settling tank.
•           These floccules or activated sludge contain large amount of metabolizing bacteria together with yeasts, fungi and protozoa.
•           Activated sludge is introduced in primary settling tank and aeration tank for rapid development of microorganisms and rapid exploitation of organic matter.
•           The process is repeated (ie., addition of settled sludge to fresh sewage – aeration – sedimentation).
•           Poor settlement of activated sludge floccules adversely affect the efficiency of sewage treatment.

•           Efficiency depends on ;

*        The ability of flocs to absorb substrates

*        Assimilation and oxidation of organic matter

*        Oxidation of nitrogen

*        Maintenance of good flocculation

•           Microorganisms found are ;

*        Enterobacter sp.

*        Corynebacterium sp.

*        Zooglea sp.

*        Thiothrix sp.

*        Cladosporium sp. etc.

d.    Anaerobic digesters

•           These digesters are used only for processing of settled sewage sludge and the treatment of very high biological oxygen demand industrial effluents.
•          Anaerobic digesters are large fermentation tanks designed to operate anaerobically with continuous supply of untreated sludge and removal of final stabilized sludge product.
•           Process involves ;

*        Mechanical mixing

*        Heating

*        Gas collection

*        Sludge addition

*        Removal of final sludge

•           3 steps of anaerobic digestion ;

a)    Fermentation

*        Fermentation of sludge cause formation of organic acids (including acetates) from organic polymers – by species such as Clostridium, Pepetostreptococcus, Eubacteria etc.

*        Acids are –butyrate, propionate, lactate, succinate, acetate, with ethanol, H₂, CO₂ etc.

b)   Acetogenesis

*        Acid products of fermentation utilized as substrates by several acetogenic bacteria–Syntrophomonas sp., Syntrophobacter sp., Acetobacteriumsp. etc.

*        Products are – acetate, H₂, CO₂etc.

c)    Methanogenesis

*        Products of acetogenesis utilized by methanogenic bacteria.

*        Acetate used and produce methane and CO₂.

*        Organisms – Methanosarcina sp., Methanobrevibacter sp., Methanothrix sp., Methanogenium sp., Methanospirillum sp. etc.

*        A balance between oxidants and reductants is maintained during methanogenic processes.

3.    Tertiary treatment

•           Chemical removal of heavy metals, nitrates, and phosphates occurs. The salts of nitrogen and phosphorous must be removed because they cause eutrophication.
•           Non biodegradable organic pollutants such as chlorophenols, polychlorinated biphenols and other synthetic pollutants are also removed.
•           Phosphorous – removed by adding lime, precipitating calcium phosphate.
•           Nitrogen – removed by volatilization as ammonia
•           Ammoniacal nitrogen can be removed by break point chlorination by adding hypochlorous acid in 1:1 ratio. This lowers biological oxygen demand because nitrification consume oxygen dissolved in the remaining water.
•           Tertiary treatment also include :

a.    Chlorination

•           Chlorine kills pathogenic bacteria and less effective on spores and certain viruses
•           Chlorine,

*        Oxidizes Fe, Mn and H₂S

*        Destroys some taste and odor producing constituents

*        Controls algae and slime organisms

*        Aids coagulation

Action of chlorine :

*        Chlorine form hydrochloric acid and hypochlorous acid when react with water, of which hydrochloric acid is neutralized by the alkalinity of water and hypochlorous acid ionizes to form hydrogen ions and hypochlorite ions.

*        Hypochlorous acid is responsible for the disinfecting action of chlorine and to a small extent due to hypochlorite ions.

•           Chlorine acts best when pH of water is around 7.
•           For disinfecting large bodies of water chlorine is applied as chlorine gas, chloramines and perchloron.
•           Chlorine demand (it is the difference between the amount of chlorine added to the water, and the amount of residual chlorine remaining at the end of a specific period of contact, at a given temperature and pH of water) of water should be estimated before chlorination.
•           Breakpoint chlorination : in this chlorine is added until the organic matter present in the water is completely oxidized, and there remains a small quantity of free chlorine. The point at which free chlorine begins to appear is known as ‘breakpoint’.

-          Super chlorination :

*        addition of large doses of chlorine

*        applicable to heavily polluted water

*        water quality may fluctuate

*        excess chlorine may be removed using SO₂after specified contact time.

b.    Ozonization

•           Ozone is produced by passing high voltage current through dry air using cylindrical electrodes made up of stainless steel or aluminium.
•           Solubility of ozone in water is higher than oxygen.
•           Ozone gas is unstable but powerful oxidizing agent.

-          Ozone is ;

*        Blue – gaseous state

*        Dark blue – liquid state

*        Black – solid state.

•           Ozonization eliminates undesirable odour, taste and colour, removes all chlorine demand from the water and also has virucidal effect.

Drawbacks :

*        the gas decomposes and disappears after its work

*        it has no residual germicidal effect

*        highly expensive.