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Identification Of Bacteria:- PDF



Once a bacterium has been obtained in a pure culture, it has to be identified. There are
different techniques for Identification of bacteria, out of those techniques staining technique
is one of them.
     Simple staining is a method of staining in which bacteria are stained by using a single
     Simple staining is also called as monochrome staining or positive staining.
     Examples of simple stain are Methylene blue, Safranin, Malachite green, Basic
        fuchsin and crystal violet etc. In simple staining procedure cell are uniformly stained.
    1. A clean grease free slide is taken .A grease free slide is made by first washing the
        slide with detergent wiping the excess water and the slide is passed through flame.
    2. On these grease free slide smear is made by using a sterile wireloop and cell
    3. These slide is allowed to air dry.
    4. After air drying these slide is rapidly passed through a flame for three to four times
        for heat fixation.
    5. After heat fixation the slide is placed on the staining rack and flooded with a
        particular stain and this stain is allowed to react for three minutes.
    6. Further the slide is washed under running water.
    7. The slide is air dried and washed under oil immersion.
                      SIMPLE STAINING PROCEDURE
                           Flow chart of Simple staining procedu
                             Take a clean grease free slide

                               Prepare a smear on slide

                              Air dry and heat fix the slide

                              Flood the slide with the stain

                          Allow the stain to react for 3 minutes

                          Wash the slide under running water

                                     Air dry the slide

                              Observe under oil immersion

                          Flow chart of simple staining procedure

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     A stain has ability to bind a cellular component. These abilities depend upon the
      charges present on cellular component and charges present on chromophore group of
    Bacteria have large number of carboxyl group on its surface and these carboxyl group
      have negative charge.
    When these carboxyl group carry out ionization reaction it shows COO– and H+
    That is COOH = COO– + H+.
    In nature these H+ ions are present on cell surface and further replaced by other
      positively charged ions like Na+ or k+.
    Now when these simple stains are used it has chloride group
    Further these stain carry out dissociation for example if the stain is Malachite green it
      will carry out dissociation and give free radicals
    That is MgCl = Mg+ and Cl–
    Now these free Mg + ions give positive charge on chromophore group.
    When these stain is applied to a cell these positively charged Mg + ions replace the
      K + or Na + present on cell surface.
    Thus a ionic bond is formed in between positively charged Mg + ions and cell
    Thus it results in staining of cell.
    Simple staining procedure stains bacteria easily and helps in observation under
    It is useful in preliminary studies of morphological characters of cell that is its size,
      shape and arrangement.[1]
    Gram staining procedure was discovered by Han’s Christian Gram in 1884.
    Gram staining is a universal staining technique used for identification and
      classification of organisms.
    In this staining, method bacteria are classified into two groups that are-
                                             1. Gram-positive bacteria
                                             2. Gram-negative bacteria
    This classification of bacteria depends upon the property of a cell to retain or lose the
      primary stain after the treatment of decolorizing agent.
    Gram staining is a basic and widely used technique.
    This technique was modified by many scientists but the best result was obtained by
      Hucker and Conn’s modification.
   1. A clean grease free slide.
   2. Bacterial cell suspension.
   3. Nichrome Wire loop.
   4. Primary stain - Crystal violet.
   5. Mordant- Gram’s Iodine.
   6. Decolorizing agent - 95% alcohol ( 95% Ethanol).
   7. Counterstain- Basic fuschin or Safranin.
   1. Take a clean grease free slide.
   2. Prepare a smear from a bacterial cell suspension on a slide by using nichrome wire
   3. Air dry and heat fix the smear.

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4. Flood the smear with a primary stain that is Crystal violet and allow it to react for 1-2
5. After Crystal violet treatment water wash treatment is given to the slide.
6. Further, the smear is treated with the mordant that is Gram’s Iodine for 1-2 minutes.
7. Excess Gram’s Iodine is removed and the slide is further treated with a decolorizing
   agent that is 95 % Ethanol.
8. After Ethanol treatment the smear is water washed and flooded with counter stain that
   is Basic fuchsin or Safranin for 1-2 minutes.
 9. Finally, the slide is washed with water, air dried and observed under oil immersion.
                             GRAM STAINING PROCEDURE

                            Take a clean grease free slide

                     Prepare a smear by using nicrome wire loop

                             Air dry and heat fix the slide

                  Flood the smear with crystal violet for 1-2 minutes

                              Wash the slide with water

                  Flood the smear with grams iodine for 1-2 minutes

                        Treat the slide with decolorizing agent

                              Wash the slide with water

                  Flood the smear with Basic fuchsin or safranin for
                                   1-2 minutes

                              Wash the slide with water

                             Observe under oil immersion
                      Flow chart of Gram staining Procedure

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      1. Cells stained with crystal violet appear violet color are Gram-positive cells.
      2. Cells stained with counter stain i.e Basic fuchsin or safranin appear pink in colour
           are Gram-negative cells.
    1. Crystal violet – It is a primary stain and a basic dye it stains all micro-organisms.
    2. Gram’s Iodine – Gram’s Iodine acts as a mordant and it forms a complex with crystal
        violet that is CV-I complex. This complex increases affinity between cell and stain.
    3. 95% Alcohol (95% Ethanol) – It is a decolorizing agent as well as a lipid solvent. It
        tries to decolorize the cell by removing the CV-I complex from the cell.
    4. Basic fuschsin or Safranin – It acts as a counterstain. It stains the cells that are
        decolorized by alcohol. Only Gram-negative bacteria get decolorize and this counter
        stain gives pink color to these cells.
    1. When a smear is stained with crystal violet it stains all cells to violet color.
    2. After application of Gram’s Iodine, its molecules acts as a mordant and forms a
        crystal violet – Gram’s Iodine complex that is CV-I complex.
    3. After CV – I complex formation this smear is subjected to decolorizing treatment by
        using 95% Ethanol for 30 seconds.
    4. The gram-positive cell has some special features due to which CV – I complex is
        unable to come outside the cell they are-
     The gram-positive cell has 1 to 4 % of lipid content due to low lipid content the cell
        get dehydrated by alcohol treatment and its pore size decreases so CV – I complex is
        trapped inside the cell.
     Peptidoglycan layer account about 40 to 90% of the dry weight of Gram-positive cell
        so due to extremely dense cross-linkage CV – I complex is trapped inside the cell.
     The         gram-positive     cell    contains    Magnesium       ribonucleate      so    this
        compound Magnesium ribonuclease molecule forms a covalent bond with CV – I
        complex and thus it doesn’t allow CV – I complex to come outside the cell.
     The gram-negative cell contains 11 to 20 % of lipid content when Gram-negative cells
        are suspended in alcohol it dissolves the lipid and thus CV – I complex comes out.
     Peptidoglycan content in Gram-negative cell wall is 5 to 10 % so due to less amount
        of cross-linkage CV – I complex comes out easily.
     Gram-negative cell lacks Magnesium ribonucleate molecules so CV – I complex is
        extracted easily from the cell.
7. The cells which get decolourised by alcohol take the counterstain and appear pink in color
these cells are Gram-negative cells.
6. After decolourisation treatment, the smear is treated with counterstain i.e Basic fuschin
and Safranin.
     Gram staining is a basic technique used for identification and classification of the
        It is a useful technique in the diagnosis of the causative agent of a clinical infection.
     It is also helpful in studying morphological characters of cells.
    1. Gram positive bacteria – Bacillus,                  Staphylococcus,         Streptococcus,
        Micrococcus etc.
    2. Gram negative bacteria – Pseudomonas, E.coli, Salmonella, Shigella, Proteus,
        Xanthomonas [2]

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    In nature, there is a variety of micro-organism each micro-organism have some
     special characters.
    Most of the microorganisms are easily stained by simple staining procedures.
    But there is some micro-organism that is not easily stained by this technique because
     they have a waxy covering on its surface. If anyhow they get stained they don’t get
     decolorize even by strong acid.
    Such organism requires a special staining technique.
    Acid-fast staining technique is a differential staining technique in bacteriology.
    This staining technique was discovered by scientist Paul Ehrlich in 1883.
    Acid-fast staining technique helps us to differentiate the organism as acid-fast and
     non-acid fast organisms.
    For staining such organism Ziehl- Neelsen staining method is used. It is also called as
     Acid-fast staining method

  1. Acid-fast organism- The organism that get stained by acid-fast staining technique but
     don’t get decolorized even by strong acid are called as an acid-fast organism.
  2. Non-acid-fast organism- The organism that easily gets stained by a staining procedure
     as well as decolorizes easily by a strong acid are a non-acid fast organism.

  1. A clean grease free slide.
  2. A bacterial cell suspension.
  3. Staining agent- Ziehl Neelsen, carbol fuchsin.
  4. Boiling water bath.
  5. Decolorizing agent – Acid alcohol.
  6. Counterstain – 1% Malachite green or 0.3 % Methylene blue.

  1. Take a clean grease free slide and prepare a smear using nichrome wire loop.
  2. Air dry and heat fix the slide.
  3. The slide is flooded with ZNCF stain and placed on a boiling water bath for steaming
     for about 3-5 minutes.
  4. During steaming the stain is repeatedly added on the slide to avoid drying of smear.
  5. Further, the slide is treated to the decolorizing agent that is acid alcohol until the stain
     disappears in washing.
  6. After decolourisation, the slide is given a water wash treatment.
  7. Further, the smear is flooded with the counterstain that is 1% Malachite green or 0.3
     % Methylene blue for about 2 minutes.
  8. After 2 minutes the slide is washed with water, air dried and observed under oil
     immersion objective.

                                                                                         Page 6
                     Flowchart of the Acid Fast staining procedure


     It is a primary stain.
     Many acid-fast bacteria are not stained with the common stain like carbol fuchsin
      because they are prepared in aqueous solution.
     These acid fast bacteria require a stain that is prepared in phenolic stain and ZNCF
      stain is prepared in phenolic solution.
     As these acid-fast bacteria have a waxy covering on their surface and phenolic stain
      solubilizes waxy covering and stains the cell.
     The cells stained with ZNCF appear pink in color.

     It is the decolorizing agent.
     It is prepared from the combination of acid that is 3% hydrochloric acid and alcohol
      that is 95% ethanol.

    It acts as a counter stain.
    It stains the decolorized cell and these cells appear green or blue in colour.

  1. Acid-fast bacteria have a waxy covering on its surface or we can say it has high lipid
     content in the cell wall.
  2. The cell wall of acid-fast bacteria is made up of lipids like Mycolic acid and
  3. Due to these high lipid content in the cell wall, these cell wall has less permeability.
  4. So first it is necessary to increase the permeability of the cell wall so the stain can
     easily penetrate in the cell.
  5. The permeability of the cell wall is increased by using phenolic solution and

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  6. After the permeability of the cell increases the cells get the stain.
  7. Now once the acid-fast bacteria get stained it doesn’t decolorize even by the strong
     decolorizing agent and appear pink in color.
  8. But the non-acid fast bacteria get decolorize easily and get stained by counter stain
     and appear green or blue in color.
  9. If we use Malachite green stain cells get the stain and appear green in color and if we
     use Methylene blue stain cells get the stain and appear blue in color.
  1. Acid-fast bacteria appear pink in color.
  2. Non-acid fast bacteria appear green or blue in color.
  1. The permeability of acid-fast cell is increased by phenolic stain because phenolic stain
     have high affinity towards the waxy covering and it is more soluble in waxy covering.
  2. For increasing the permeability we use heat steaming because steaming softens the
     waxy material and allow easy penetration of stain.
  1. Acid-fast staining is useful in the diagnosis of Tuberculosis and leprosy.[3]

                                                                                     Page 8
                         BIOCHEMICAL TESTS
 Many biochemical tests are performed for identification of bacteria, Out of which IMViC
tests is very important.
IMViC Tests
Each of the letters in “IMViC” stands for one of these tests. “I” is for indole; ”M”is for
methyl red;”V” is for Voges-Proskauer, and “C” is for citrate, lowercase “I” is added for the
ease of pronunciation. “IMViC” is an acronym that stands for four different tests.[4]



This is tested in a peptone water culture after 48 or 96 hours incubation at 37°C.This test
demonstrates the production of indole from tryptophan. Add 0.5 ml Kovac’s reagent and
shake gently.

Red colour in the top of the tube indicates a positive reaction.
Kovac’s reagent consists of
Paradimethylaminobenzaldehyde ………10gm
Amyl or isoamyl alcohol………………...150ml
Concentrated HCL……………………….50ml
This is prepared in small quantities and stored in the refrigerator.

This test is employed to detect the production of acid during the fermentation of glucose and
the maintenance of pH below 4.5 in an old culture. Five drops of 0.04% solution of methyl
red are added to the culture in glucose phosphate medium which had been incubated at 30°C
for five days, mixed well and read at once. Red color is positive while yellow signifies a
negative test.

This test depends on the production of acetyl methyl carbinol from pyruvic acid, as an
intermediate stage in its conversion to 2:3 butylene glycol. In the presence of alkali and
atmospheric oxygen, the small amount of alkylmethyl carbinol present in the medium is
oxidized to diacetyl which reacts with the peptone of the broth to give a red colour.
The test is performed by adding 0.6 ml of a 5% solution of α-napthol in ethanol and 0.2 ml of
40% KOH to one ml of a glucose phosphate medium culture of the organism incubated at
30°C for five days or 37°C for 48 hours. In a positive, a pink colour appears in 2-5 minutes,
depening to magenta or crimson in half an hour. Traces of pink colouration should be

Kosers citrate medium has citrate as the sole source of carbon. Ability to use this substance is
indicated by the production of turbidity of the medium.
Indole, MR, VP and citrate tests are very useful in the identification and classification of
enteric Gram negative bacteria. [5]

                                                                                         Page 9
    Sterilization is the complete removal of microorganisms from an object or surfaces.
    Sterilization is obtained when microorganisms are subjected to antimicrobial agents
     for sufficient time and at optimum conditions.
  Some physical methods associated with sterilization are explained below
    Heat sterilization is the most effective and widely used method of sterilization, where
     the bactericidal activity results through the destruction of enzymes and other essential
     cell constituents.
    The effects of heat sterilization occur more rapidly in a fully hydrated state, as it
     requires a lower heat input, with low temperature and less time, under high humidity
     conditions where the denaturation and hydrolysis reactions are predominant, rather
     than in the dry state where oxidative changes take place.
    Under circumstances where thermal degradation of a product is possible, it can
     usually be minimized by adopting a higher temperature range, as the shorter exposure
     times generally result in a lower partial degradation.
    This method of sterilization is applicable to thermostable products. Still, it can be
     applied to both moisture-sensitive and moisture-resistant products, for which dry
     (160–180°C) and moist (121–134°C) heat sterilization procedures are respectively
    Dry sterilization is the process of removing microorganisms by applying moisture-
     free heat which is appropriate for moisture-sensitive substances.
    The dry heat sterilization process is based on the principle of conduction; that is the
     heat is absorbed by the outer surface of an item and then passed onward to the next
     layer. Ultimately, the entire item reaches the proper temperature needed to achieve
    Dry moisture-less heat destroys microorganisms by causing denaturation of proteins
     and also lyses the proteins in many organisms, causes oxidative free radical damage,
     causes drying of cells, and can even burn them to ashes, as in incineration
    Dry heat sterilization is used for the sterilization of materials which are difficult to
     sterilize by moist heat sterilization for several reasons.
    Substances like oil, powder, and related products cannot be sterilized by moist heat
     because moisture cannot penetrate into deeper parts of oily materials, and powders are
     destroyed by moisture.
    Similarly, laboratory equipment like Petridishes and pipettes are challenging to
     sterilize by moist heat due to the penetration problem.
    The lethal effects of dry heat on microorganisms are primarily due to oxidative
     processes which are less effective when compared to the hydrolytic damage that
     results from exposure to steam in moist heat sterilization.
    Thus, in dry heat sterilization usually higher temperatures in the range 160–180°C are
     employed and also require exposure times of up to 2 hours depending upon the
     temperature employed.
    This principle is used in instruments like hot air oven and incineration, which
     generates very hot moisture-free air.
    The primary industrial application of dry heat sterilization is in the sterilization of
     glass bottles which are to be filled aseptically.

                                                                                    Page 10
      In addition to the fact that this method achieves an adequate sterility assurance level,
       this method also destroys bacterial endotoxins (which are the products of Gram-
       negative bacteria also called pyrogens, which cause fever when injected into the
       body) which are difficult to eliminate through other sterilization techniques.
      For the purposes of depyrogenation of glass, temperatures of approximately 250°C
       are used.
      There are different types of dry heat sterilization which are explained below:

Table 1:(Temperature time relationship in hot air oven)

               The most common time –temperature relationships for sterilization with hot
       sterilizers are
               170°C(340°F) for minutes,
               160°C(320°F) for 60 minutes, and
               150°C(300°F) for 150 minutes or longer depending up the volume

   Red heat sterilization is the process of instant sterilization by holding the instruments
     in a Bunsen flame till they become red hot.
   This method is based on dry heat sterilization is commonly used for sterilization of
     instruments like incubation loops, wires, and points of forceps.
   This process ensures effective sterilization; however, it is only limited to substances
     that can endure heating until redness in flame.

   Flaming is a type of dry sterilization that involves exposure of metallic objects to
    flame for some time where the flame burns microbes and other dust presents in the
   In the case of flaming, the instrument is dipped in alcohol or spirit before burning it in
    a gas flame.
   This process doesn’t ensure sterility and is not as effective as red heat sterilization.

   Incineration is the process of sterilization along with a significant reduction in the
     volume of the wastes.
   It is usually conducted during the final disposal of the hospital or other residues.
   The scraps are heated till they become ash which is then disposed of later.
   This process is conducted in a device called incinerator.

   Hot air oven is a method of dry heat sterilization which allows the sterilization of
     objects that cannot be sterilized by moist heat.
   It uses the principle of conduction in which the heat is first absorbed by the outer
     surface and is then passed into the inner layer.
   A hot air oven consists of an insulated chamber that contains a fan, thermocouples,
     temperature sensor, shelves and door locking controls.
   The commonly-used temperatures and time that hot air ovens need to sterilize
     materials are 170°C for 30 minutes, 160°C for 60 minutes, and 150°C for 150

                                                                                      Page 11
      These ovens have applications in the sterilization of glassware, Petri plates, and even
       powder samples.

       Fig.1:(HOT AIR OVEN)

     Moist heat sterilization is one of the most effective method of sterilization where the
       steam under pressure acts as a bactericidal agent.
     Moist heat sterilization usually involves the use of steam at temperatures in the range
    High pressure increases the boiling point of water and thus helps achieve a higher
      temperature for sterilization.
    High pressure also facilitates the rapid penetration of heat into deeper parts of
      material and moisture present in the steam causes the coagulation of proteins causing
      an irreversible loss of function and activity of microbes.
    The high temperature-short time cycles not only often result in lower fractional
      degradation, but they also provide the advantage of achieving higher levels of sterility
      assurance due to more significant inactivation factors.
    The most commonly used standard temperature-time cycles for clinical porous
      specimens (e.g. surgical dressings) and bottled fluids are 134°C for 3 minutes and
      121°C for 15 minutes, respectively.
    An autoclave is a device that works on the principle of moist heat sterilization through
      the generation of steam under pressure.

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      In this method, the microorganisms are killed by coagulating their proteins, and this
       method is much more effective than dry heat sterilization where microbes are killed
       through oxidation.
      In the pharmaceutical and medical sectors, it is used in the sterilization of dressings,
       sheets, surgical and diagnostic equipment, containers, and aqueous injections,
       ophthalmic preparations, and irrigation fluids, in addition to the processing of soiled
       and contaminated items.
      Moist heat can be used in sterilization at different temperatures:

   The sterilization technique employed at a temperature below 100°C involves
   In this process, all non-spore forming microbes are killed in milk by subjecting the
     milk to a temperature of 63°C for 30 minutes (the holder method) or 73°C for 20
     seconds (the flash method).
   In pasteurization, however, not all the pathogenic organisms are killed. The principle
     of pasteurization is the logarithmic reduction in the number of viable microbes so that
     they can no longer cause diseases.
   All mesophilic non-sporing bacteria can be killed by exposure to a moist heat at 60°C
     for half an hour with the exception of some organisms which require different
     temperature-time cycles.
   The milk is not heated above its boiling point as the milk might curdle, and its
     nutritional value might be destroyed.
   Besides milk, other fluids and equipment like vaccines of non-sporing bacteria are
     also pasteurized at 60°C for 1 hour in special water baths.
   Similarly, serum and body fluids with congealable proteins are also sterilized at 56°C
     for 1 hour in water baths.

    Boiling at 100°C is a moist heat sterilization technique that doesn’t ensure complete
      sterility, but is enough for the removal of pathogenic vegetative microbes and some
    In this case, the items to be sterilized are immersed in boiling distilled water for 30-40
    Distilled water is preferred because hard water might result in the formation of a film
      of calcium salts on the instruments.
    Tyndallization is a method that is used for sterilization of media with sugar and
      gelatin at 100°C for 30 minutes on three successive days so as to preserve sugar
      which might be decomposed at a higher temperature.
    Moist heat at 100°C is applicable for contaminated dishes, beddings, pipettes, and
      other instruments that are not soiled or contaminated as well as for objects that are
      temperature sensitive.

    Moist heat sterilization above 100°C involves sterilization by steam under pressure.
    Water usually boils at 100°C under normal atmospheric pressure (760 mm of Hg);
     however, the boiling point of water increases if the pressure is to be increased.
    This principle is employed in an autoclave where the water boils at 121°C at the
     pressure of 15 psi or 775 mm of Hg.

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   As a result, the steam under pressure has a higher penetrating power. When this steam
    comes in contact on the surface, it kills the microbes by giving off latent heat.
   The condensed liquid ensures the moist killing of the microbes.
   Autoclaves are used for the sterilization of contaminated instruments along with
    different culture media as it ensures complete sterility.


          Table 2:(Pressure temperature relationship in autoclave)

                         Pressure temperature relations in
                     Pressure in Temperature Temperature
                         psi          in °C           in °F
                          5            109             228
                         10            115             240
                         15            121             250
                         20            126             259
                         25            130             267
                         30            135             275

                                                                                Page 14
Table 3:(Heat sterilization method, its mechanism, merits, demerits & applications)

Sl    Method         Mechanis     Merits             Demerits       Applications
no                   m
1     Heat            Destroys    Most widely         Can      be   Dry heat is applicable for
      sterilizatio   bacterial    used        and    applied only   sterilizing glasswares and
      n              endotoxins   reliable           to       the   metal surgical instruments
                                  method        of   thermastabl    and moist heat is the most
                                  sterilization      e products     dependable     method    for
                                  involving                         decontamination           of
                                  destruction of                    laboratory waste and the
                                  enzymes and                       sterilization of laboratory
                                  other                             glassware,     media    and
                                  essential cell                    reagents.

    Irradiation is the process of exposing surfaces and objects to different kinds of
     radiation for sterilization.
    Mainly electromagnetic radiation is used for sterilization.
    The major target for these radiations is considered to be microbial DNA, where
     damage occurs as a result of ionization and free radical production (gamma-rays and
     electrons) or excitation (UV light).

    Infrared radiation (IR) is a method of thermal sterilization in which the radiation is
     absorbed and then converted into heat energy.
    For this purpose, a tunnel containing an IR source is used. The instruments and
     glassware to be sterilized are kept in a tray are then passed through the tunnel on a
     conveyer belt, moving at a controlled speed.
    During this movement, the instruments will be exposed to the radiation, which will
     result in a temperature of about 180°C for about 17 minutes.
    IR is applicable for mass sterilization of packaged items like syringes and catheters.

   Ultraviolet radiation includes light rays from 150-3900 Å, of which 2600 Å has the
    highest bactericidal effect.
   Non-ionizing waves have a very little penetration power, so microorganisms only on
    the surface are killed.
   Upon exposure, these waves are absorbed by many materials, particularly nucleic
   The waves, as a result, cause the formation of pyrimidine dimers which bring error in
    DNA replication and cause the death of microbes by mutation.
   UV radiation owing to its poor penetrability of conventional packaging materials is
    unsuitable for sterilization of pharmaceutical dosage forms.

                                                                                       Page 15
      It is, however, applied in the sterilization of air, for the surface sterilization of aseptic
       work areas, and the treatment of manufacturing-grade water.

     X-ray and gamma rays are the commonly used ionizing radiation for sterilization.
     These are high energy radiation which causes ionization of various substances along
      with water.
     The ionization results in the formation of a large number of toxic O2 metabolites like
      hydroxyl radical, superoxide ion, and H2O2 through ionization of water.
     These metabolites are highly oxidizing agents and kill microorganisms by oxidizing
      various cellular components.
     With ionizing radiation, microbial resistance decreases with the presence of moisture
      or dissolved oxygen (as a result of increased free radical production) and also with
      elevated temperatures.
     Radiation sterilization is generally exposed to items in the dried state which include
      surgical instruments, sutures, prostheses, unit-dose ointments, plastic syringes, and
      dry pharmaceutical products.

   The process of filtration is unique among sterilization techniques in that it removes,
     rather than destroys, microorganisms.
   Further, it is capable of preventing the passage of both viable and nonviable particles
     and can thus be used for both the clarification and sterilization of liquids and gases.
   The primary mechanisms involved in filtration are sieving, adsorption, and trapping
     within the matrix of the filter material.
   Filtration uses membranous filters that have tiny pores that let the liquid pass through
     but prevent bigger particles such as bacteria from passing through the filter.
     Therefore, the smaller the pore, the more likely the filter is to stop more things from
     going through it.
   Certain types of filter (membrane filters) also have an essential role in sterility testing,
     where they can be employed to trap and concentrate contaminating organisms from
     solutions under test.
   These filters are then placed in a liquid nutrient medium and incubated to encourage
     growth and turbidity.
   The principal application of sterilizing-grade filters is the treatment of heat-sensitive
     injections and ophthalmic solutions, biological products, air, and other gases for
     supply to aseptic areas.
   They may also be required in industrial applications where they become part of
     venting systems on fermenters, centrifuges, autoclaves, and freeze dryers.

    Membrane filters, in the form of discs, can be assembled into pressure-operated filter
     holders for syringe mounting and in-line use or vacuum filtration tower devices for
     filtration of liquid.
    Filtration under pressure is generally considered most suitable, as filling at high flow
     rates directly into the final containers is possible without problems of foaming,
     solvent evaporation, or air leaks.
    Membrane filters are often used in combination with a coarse-grade fiberglass depth
     profiler to improve their dirt-handling capacity.

                                                                                          Page 16
    Filters employed for this generally consist of pleated sheets of glass microfibres
     separated and supported by corrugated sheets of Kraft paper or aluminum which are
     employed in ducts, wall or ceiling panels, or laminar air flow cabinets.
    These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of
     particles >0.3mm in diameter and thus are acting as depth filters.
    In practice, their microorganism removal efficiency is rather better as the majority of
     bacteria are found associated with dust particles.
    Other applications of filters include sterilization of venting or displacement air in
     tissue and microbiological culture (carbon filters and hydrophobic membrane filters);
     decontamination of air in mechanical ventilators (glass fiber filters); treatment of
     exhausting air from microbiological safety cabinets (HEPA filters); and the
     clarification and sterilization of medical gases (glass wool depth filters and
     hydrophobic membrane filters).[6]

Table 4:(Filtration method, its mechanism, merits, demerits & applications)

     Sl     Method       Mechanism      Merits               Demerits          Applications
     1      Filtration   Does    not    It is used for       Does        not   In      this     method
                         destroy but    both           the   differentiate     sterilizing grade filters
                         remove the     clarification and    between           are used in the
                         microorgani    sterilization of     viable     and    treatment     of    heat
                         sms            liquids       and    non     viable    sensitive     injections
                                        gases as it is       particles         and           opthalmic
                                        capable         of                     solutions, biological
                                        preventing the                         products and air and
                                        passage of both                        other for supply to
                                        vible and non                          asceptic areas
                                        viable particles

                             PHYSICAL STERILISATION

             HEAT                       IRRDIATION                                   FILTRATION


                          Classification of physical sterilization

                                                                                        Page 17

      Chemical Sterilization is the process of removal of microorganisms by the use of
       chemical bactericidal agents.
      Even if physical methods of sterilization are more appropriate for effective
       sterilization, it is not always appropriate to use for heat-sensitive materials like
       plastics, fiber optics, and biological specimens.
      Under such conditions, chemical either in liquid or gaseous state can be used for
       sterilization. However, it is crucial to ensure that the materials undergoing
       sterilization are compatible with the chemical being used.
      Besides, it is important to adopt safety rules in the workplace safety during the use of
       chemical agents.
      The chemical method of sterilization can be categorized as liquid and gaseous

    Gaseous sterilization involves the process of exposing equipment or devices to
      different gases in a closed heated or pressurized chamber.
    Gaseous sterilization is a more effective technique as gases can pass through a tiny
      orifice and provide more effective results.
    Besides, gases are commonly used along with heat treatment which also facilitates the
      functioning of the gases.
    However, there is an issue of release of some toxic gases during the process which
      needs to be removed regularly from the system.
    The mechanism of action is different for different types of gases.
Some of the common gases used for gaseous sterilization are explained below

   Ethylene sterilize, pasteurize, or disinfect different types of equipment and surfaces
     because of its wide range of compatibility with different materials.
   EO treatment often replaces other sterilization techniques like heat, radiation, and
     even chemicals in cases where the objects are sensitive to these techniques.
   This method is a widespread method used for almost 70% of all sterilizations and
     around 50% for disposable medical devices.
   The mechanism of antimicrobial action of this gas is assumed to be through the
     alkylation of sulphydryl, amino, hydroxyl, and carboxyl groups on proteins and imino
     groups of nucleic acids.
   EO treatment is usually conducted at the temperature range of 30-60°C for several
     hours which aids in the activity of the gas.
   The efficacy of the gas depends on the concentration of gas available for each article
     which is greatly assisted by the good penetrating nature of the gas, which diffuses
     readily into many packaging materials including rubber, plastics, fabric, and paper.
   Ethylene oxide kills all known microorganisms, such as bacteria (including spores),
     viruses, and fungi (including yeasts and molds), and is compatible with almost all
     materials even when repeatedly applied.
   This process, however, is not without drawbacks as the level of gas in the sterilizer
     goes on decreasing due to absorption, and the treated articles need to undergo a
     process of desorption to remove the toxic residual wastes

                                                                                       Page 18
        Organisms are more resistant to ethylene oxide treatment in a dried state, as are those
         protected from the gas by inclusion in crystalline or dried organic deposits.

Table 5: (Gaseous & Radiation sterilization methods, mechanism, merits, demerits &

Sl no.    Method          Mechanism Merits           Demerits                Applications
1         Gaseous         Alkylation Penetrating     Gases           being   Ethylene oxide
          sterilization              ability of      alkylating     agents   gas has been used
                                     gases           and       potentially   widely to process
                                                     mutagenic         and   heat sensitive
                                                     carcinogenic            devices
 2        Radiation       Ionization   It is a       Undesirable             Radiation
          sterilization   of nucleic   useful        changes occur in        Sterilization is
                          acids        method for irradiated products,       generally applied
                                       the           an                      to articles in the
                                       industrial    example is aqueous      dry state;
                                       sterilization solution where          including surgical
                                       of heat       radiolysis of water     instruments,
                                       sensitive     occurs.                 sutures,
                                       products                              Prosthesis,
                                                                             unit doses.

   Formaldehyde is another important highly reactive gas which is used for sterilization.
   This gas is obtained by heating formalin (37%w/v) to a temperature of 70-80°C.
   It possesses broad-spectrum biocidal activity and has found application in the
    sterilization of reusable surgical instruments, specific medical, diagnostic and
    electrical equipment, and the surface sterilization of powders.
   Formaldehyde doesn’t have the same penetrating power of ethylene oxide but works
    on the same principle of modification of protein and nucleic acid.
   As a result of the low penetrating power, its use is often limited to paper and cotton
   Formaldehyde can generally be detected by smell at concentrations lower than those
    permitted in the atmosphere and thus can be detected during leakage or other such

    Nitrogen dioxide is a rapid and effective sterilant that can be used for the removal of
     common bacteria, fungi, and even spores.
    NO2 has a low boiling point (20°C) which allows a high vapor pressure at standard
    This property of NO2 enables the use of the gas at standard temperature and pressure.
    The biocidal action of this gas involves the degradation of DNA by the nitration of
     phosphate backbone, which results in lethal effects on the exposed organism as it
     absorbs NO2.

                                                                                        Page 19
      An advantage of this gas is that no condensation of the gas occurs on the surface of
       the devices because of the low level of gas used and the high vapor pressure. This
       avoids the need for direct aeration after the process of sterilization.
      Nitrogen dioxide is a rapid and effective sterilant that can be used for the removal of
       common bacteria, fungi, and even spores.
      NO2 has a low boiling point (20°C) which allows a high vapor pressure at standard
      This property of NO2 enables the use of the gas at standard temperature and pressure.
      The biocidal action of this gas involves the degradation of DNA by the nitration of
       phosphate backbone, which results in lethal effects on the exposed organism as it
       absorbs NO2.
      An advantage of this gas is that no condensation of the gas occurs on the surface of
       the devices because of the low level of gas used and the high vapor pressure. This
       avoids the need for direct aeration after the process of sterilization.

   Ozone is a highly reactive industrial gas that is commonly used to sterilize air and
    water and as a disinfectant for surfaces.
   Ozone is a potent oxidizing property that is capable of destroying a wide range of
    organisms including prions, without the use of hazardous chemicals as ozone is
    usually generated from medical-grade oxygen.
   Similarly, the high reactivity of ozone allows the removal of waste ozone by
    converting the ozone into oxygen by passing it through a simple catalyst.
   However, because ozone is an unstable and reactive gas, it has to be produced on-site,
    which limits the use of ozone in different settings.
   It is also very hazardous and thus only be used at a concentration of 5ppm, which is
    160 times less than that of ethylene oxide.

     Liquid sterilization is the process of sterilization which involves the submerging of
       equipment in the liquid sterilant to kill all viable microorganisms and their spores.
     Although liquid sterilization is not as effective as gaseous sterilization, it is
       appropriate in conditions where a low level of contamination is present.
Different liquid chemicals used for liquid sterilization includes the following

   Hydrogen peroxide is a liquid chemical sterilizing agent which is a strong oxidant and
    can destroy a wide range of microorganisms.
   It is useful in the sterilization of heat or temperature-sensitive equipment like
    endoscopes. In medical applications, a higher concentration (35-90%) is used.
   H2O2 has a short sterilization cycle time as these cycles are as short as 28 minutes
    where ethylene oxide has cycles that as long as 10-12 hours.
   However, hydrogen peroxide has drawbacks like low material compatibility, lower
    capacity of penetration, and associated health risks.
   Vaporized hydrogen peroxide (VHP) is used to sterilize largely enclosed and sealed
    areas, such as entire rooms and aircraft interiors.

                                                                                     Page 20
   Glutaraldehyde is an accepted liquid sterilizing agent which requires comparatively
     long immersion time. For the removal of all spores, it requires as long as 22 hours of
     immersion time.
   The presence of solid particles further increases the immersion time.
   The penetration power is also meager as it takes hours to penetrate a block of tissues.
   The use of glutaraldehyde is thus limited to certain surfaces with less contamination

   Hypochlorite solution, which is also called liquid bleach, is another liquid chemical
    that can be used as a disinfectant, even though sterilization is difficult to obtain with
    this chemical.
   Submerging devices for a short period in liquid bleach might kill some pathogenic
    organisms but to reach sterilization submersion for 20-24 hours is required.
   It is an oxidizing agent and thus acts by oxidizing organic compounds which results in
    the modification of proteins in microbes which might ultimately lead to death.
   Appropriate concentrations of hypochlorite can be used for the disinfection of
    workstations and even surfaces to clean blood spills and other liquids.[7]

                          CHEMICAL STERILIZATION

GASEOUS STERILIZATION                                           LIQUID STERILIZATION

                           Classification of chemical sterilization

Evaluation of the efficiency of sterilization methods: The term 'sterile' in a microbiological
context, means no surviving organisms, whatsoever. Thus, there are no degrees of sterility; an
item is either sterile or it is not, and so there are no level of contamination which may be
considered negligible or insignificant and therefore acceptable. True sterility, represented by
zero survivors, can only be achieved after an infinite exposure period or radiation dose.
Clearly then, it is illogical to claim, or expect, that a sterilization procedure will guarantee
sterility. Thus, the likelihood of a product being produced free of microorganisms is best
expressed in terms of the probability of an organism surviving the treatment process, a
possibility not entertained in the absolute term 'sterile'. From this approach has arisen the
concept of 'sterility assurance' or a microbial safety index which gives a numerical value to
the probability of a single surviving organism remaining to contaminate a processed product.
For pharmaceutical products, the most frequently applied standard is that the probability, post
sterilization, of a non-sterile unit is 1 in 1 million units processed (i.e., <=10 -6). The
sterilization protocol necessary to achieve this with any given organism of known D-value
(decimal reduction time) can be established from the inactivation factor (IF) which may be
defined as
                                          IF = 10 × t D
 where 't' is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing
radiation; and 'D' is D-value (time taken at a fixed temperature or the radiation dose required

                                                                                         Page 21
to achieve a 90% reduction in viable cells. (D-value is one the functions to indicate the
efficiency of sterilization process)
IF for selected sterilization protocols and their corresponding biological indicator
Moist heat (121℃ for 15 min) - B. stearothermophilus, D-value - 1.5 min, Log IF - 10;
Dry heat (160℃ for 120 min) - B. subtilis var niger, D-value - max. 3 min,
 Log IF - Min. 40;
Irradiation (25 kGy) - B. pumilus, D-value - 1.9 kGy,
Log IF - 13.2
This is the simplest method of calculating the probability of achieving sterility for any given
initial survival level.
From the above-mentioned D-values and Log IF (or IF) values, it is clear that moist heat.[8]

                               STERILITY INDICATORS
    Monitoring physical indicators involves observing the gauges or displays on the
      sterilizer and recording the time, temperature, and pressure associated with each
      sterilization cycle for each load.
    Some sterilizers have recording devices that print out these parameters.
    Correct readings do not guaranty sterilization, but incorrect readings can be the first
      indication of a problem with the sterilization cycle and suggest the load may not be
      sterile. [9]
     Chemical indicators use sensitive chemicals to assess critical variables (e.g., time,
       temperature, or steam saturation) during a sterilization cycle.
     They are applied either to the outside or placed on the inside of each instrument unit
       (e.g., packs, peel pouches, containers, etc…).
     They do not prove that sterilization has been achieved, but they can provide an early
       indication of a problem and where in the sterilization process the problem might
    Biological indicators (BIs), or spore tests, assess directly the killing of known highly
      resistant, non pathogenic bacterial spores.
    Geobacillus stearothermophilus (G. stearothermophilus) spores test steam and
      unsaturated chemical vapor sterilizers.
    Bacillus atrophaeus (B. atrophaeus) spores test dry heat sterilizers.
    Bacterial spores in the test products are more resistant and are present in greater
      numbers than common microbial contaminants found on patient-care items..[11]

                                                                                      Page 22
   Steam sterilizer
   Dry heat sterilizer
   ETO Sterilizer
   Sterilizing tunnel
   CIP System
   SIP System[12]

(A)                                           (B)


Fig. 3: (A) Steam sterilizer; (B) Dry heat sterilizer; (C) ETO Sterilizer; and (D) CIP