GE Basket microbiology (1).pptx STERILIZATION AND ALL

BASIC MICROBIOLOGY

1. GE Basket MICROBIOLOGY UNIT – 1 BACTERIA By Sujoy Tontubay, Assistant Professor (Allied Health Sciences), ILEAD, Kolkata. B.Sc. (University of Calcutta), M.Sc. in Biomedical Laboratory Science(V.U), Post Graduate Diploma in Epidemiology and Public Health(V.U) Former Guest Lecturer (Medical Laboratory technology) in Guru Nanak Institute of Pharmaceuticals Science and Technology (G.N.I.P.S.T), Lokenath Educational Institute.

2. Bacteria “Bacteria are unicellular organisms belonging to the prokaryotic group where the organisms lack a few organelles and a true nucleus”. Bacteria are microscopic, unicellular, prokaryotic organisms. They do not have membrane-bound cell organelles and lack a true  nucleus , They constitute a large domain of prokaryotic microorganisms. Bacteria  are small single-celled organisms.  Bacteria  are found almost everywhere on Earth and are vital to the planet's ecosystems. Most bacteria are  0.2μm (micron) in diameter and 2−8μm (micron) in length.

3. Bacteria Cell Structure Cell Wall  - Each bacterium is enclosed by a rigid cell wall composed of peptidoglycan, a protein-sugar (polysaccharide) molecule. The wall gives the cell its shape and surrounds the cytoplasmic membrane, protecting it from the environment. Capsule  - Some species of bacteria have a third protective covering, a capsule made up of polysaccharides (complex carbohydrates). Capsules play a number of roles, but the most important are to keep the bacterium from drying out and to protect it from phagocytosis (engulfing) by larger microorganisms. Cytoplasm  - The cytoplasm, or protoplasm, of bacterial cells is where the functions for cell growth, metabolism, and replication are carried out. It is a gel-like matrix composed of water, enzymes, nutrients, wastes, and gases and contains cell structures such as ribosomes, a chromosome, and plasmids. Ribosome: Ribosomes are microscopic "factories" found in all cells, including bacteria. It is a tiny granule made up of RNA and proteins. They are the site of protein synthesis. They are free-floating structures that help in transferring the genetic code.

4. Flagella : Flagella are long hair-like filamentous structures of about 4 – 5 μm long and 0.01 – 0.03 μm in diameter. They confer motility to the bacteria. It helps the cell move clockwise and anticlockwise forward and also helps the cell spin. The flagella beat in a propeller-like motion to help the bacterium move toward nutrients; away from toxic chemicals; or, in the case of the photosynthetic cyanobacteria; toward the light. Nucleoid  - The nucleoid is a region of cytoplasm where the chromosomal DNA is located. It is not a membrane bound nucleus, but simply an area of the cytoplasm where the strands of DNA are found. Most bacteria have a single, circular chromosome that is responsible for replication. Plasmid: Plasmids are a small circle of DNA. Bacterial cells have many plasmids. A  plasmid  is a small, extrachromosomal  DNA  molecule within a cell that is physically separated from chromosomal  DNA  and can replicate independently. Pili  - Many species of bacteria have pili (singular, pilus), small hairlike projections emerging from the outside cell surface. These outgrowths assist the bacteria in attaching to other cells and surfaces, such as teeth, intestines, and rocks. Without pili, many disease-causing bacteria lose their ability to infect because they're unable to attach to host tissue. Cytoplasmic Membrane  - A layer of phospholipids and proteins, called the cytoplasmic membrane, encloses the interior of the bacterium, regulating the flow of materials in and out of the cell.

5. Classification of Bacteria Bacteria can be classified into various categories based on their features and characteristics. The classification of bacteria is mainly based on the following: Shape Composition of the cell wall Mode of respiration Mode of nutrition

6. Classification of bacteria based on Shape Type of Classification Examples Bacillus (Rod-shaped) Escherichia coli (E. coli) Spirilla or spirochete (Spiral) Spirillum volutans Coccus (Sphere) Streptococcus pneumoniae Vibrio (Comma-shaped) Vibrio cholerae

7. Classification of bacteria based on the Composition of the Cell Wall Type of Classification Examples Peptidoglycan cell wall Gram-positive bacteria Lipopolysaccharide cell wall Gram-negative bacteria E. coli

8. Classification of bacteria based on the Mode of Nutrition Type of Classification Examples Autotrophic Bacteria Cyanobacteria Heterotrophic Bacteria All disease-causing bacteria Salmonella typhi

9. Classification of bacteria based on the Mode of Respiration Type of Classification Examples Anaerobic Bacteria Actinomyces Aerobic Bacteria Mycobacterium Clostridium perfringens (Anaerobic) Nocardia pseudobrasiliensis (Aerobic)

10. Staining reaction Staining is technique used in microscopy to enhance contrast in the microscopic image. A stain is a chemical dye which is utilized for colouring biological materials. Stains are organic compounds consisting of two components, the chromophore that imparts colour to the stain as well as the auxochrome group. There are mainly four different kind types of stains: 1. Differential stain: Chemical that can bind to different structures or organisms in different manners. Eg : Ethanol, crystal violet, Gram’s iodine. 2. Simple stain: Where the dye or stain is nonspecific and stains all the features and entities of the specimen. Eg : Safranin, methylene blue. 3. Negative stain: Type of stain that stains the background but does not stain the specimen. Eg : India ink, nigrosine. 4. Special stain: Dye that has the ability to stain particular minute subcellular structures. Eg : Biebrich Scarlat stain has the ability to stain muscles and collagen.

11. Gram Staining Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms.

12. Principle of Gram Staining When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in color. In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and appears red in color.

13. Reagents Used in Gram Staining Crystal Violet, the primary stain Iodine, the mordant A decolorizer made of acetone and alcohol (95%) Safranin, the counterstain

14. Procedure of Gram Staining Take a clean, grease free slide. Prepare the smear of suspension on the clean slide with a loopful of sample. Air dry and heat fix Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. Flood the gram’s iodine for 1 minute and wash with water. Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water. Add safranin for about 1 minute and wash with water. Air dry, Blot dry and Observe under Microscope.

15. Interpretation Gram Positive: Blue/Purple Color Gram Negative: Red Color Examples Gram Positive Bacteria: Actinomyces, Bacillus, Clostridium, Corynebacterium, Enterococcus, Gardnerella, Lactobacillus, Listeria, Mycoplasma, Nocardia, Staphylococcus, Streptococcus, Streptomyces ,etc. Gram Negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio , acetic acid bacteria, Legionella et

16. Endospore Staining In 1922,  Dorner  published a method for staining endospores.  Shaeffer and Fulton  modified Dorner’s method in 1933 to make the process faster The endospore stain is a differential stain which selectively stains bacterial endospores. The main  purpose  of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers. 

17. Principle of Endospore Staining Bacterial endospores  are metabolically inactive, highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions. The bacteria can remain in this suspended state until conditions become favorable and they can germinate and return to their vegetative state. In the Schaeffer-Fulton`s method , a primary stain- malachite green  is forced into the spore by steaming the bacterial emulsion.  Malachite green  is water soluble and has a low affinity for cellular material, so  vegetative cells  may be  decolorized  with  water .  Safranin  is then applied to counterstain any cells which have been decolorized. At the end of the staining process,  vegetative cells  will be  pink,  and  endospores  will be  dark green . Spores may be located in the  middle  of the cell, at the  end  of the cell, or  between  the  end  and  middle  of the cell. Spore shape may also be of diagnostic use. Spores may be  spherical  or  elliptical .

18. Reagents used for Endospore Staining Primary Stain: Malachite green (0.5% ( wt /vol) aqueous solution) ( 0.5 gm of malachite green 100 ml of distilled water ) Decolorizing agent Tap water or Distilled Water Counter Stain: Safranin ( Stock solution (2.5% ( wt /vol) alcoholic solution) 2.5 gm of safranin O 100 ml of 95% ethanol )

19. Procedure of Endospore Staining Take a clean grease free slide and make smear using sterile technique. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide. Saturate the blotting paper with  malachite green  stain solution and steam for  5 minutes , keeping the paper moist and adding more dye as required. Alternatively, the slide may be steamed over a container of boiling water. Wash the slide in  tap water. Counterstain with  0.5% safranin  for  30 seconds . Wash with tap water; blot dry. Examine the slide under microscope for the presence of endospores. Endospores are bright green and vegetative cells are brownish red to pink.

20. Result of Endospore Staining Endospores : Endospores are bright green. Vegetative Cells:  Vegetative cells are brownish red to pink.

21. Endospore Staining by Dorner’s Method Reagents used for Endospore Staining Carbolfuchsin stain ( 0.3 gm of basic fuchsin 10 ml of ethanol, 95% (vol/vol) 5 ml of phenol, heat-melted crystals 95 ml of distilled water ) Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water. Mix and let stand for several days. Filter before use. Decolorizing solvent (acid-alcohol) ( 97 ml of ethanol, 95% (vol/vol) 3 ml of hydrochloric acid (concentrated)) Counterstain ( Nigrosin solution) ( 10 gm of nigrosin 100 ml of distilled water)

22. Procedure Take a clean grease free slide and make smear using sterile technique. Air dry and heat fix the organism on a glass slide and cover with a square of blotting paper or toweling cut to fit the slide. Saturate the blotting paper with  carbolfuchsin  and steam for  5 to 10 minutes , keeping the paper moist and adding more dye as required. Alternatively, the slides may be steamed over a container of boiling water. Remove the blotting paper and decolorize the film with  acid-alcohol  for  1 minute ; rinse with tap water and blot dry. Further take a drop of  nigrosine  on one end of a slide and make a thin film of a stain all over the smear with the help of other slide. Allow the film of Nigrosin to air dry. After air drying observe the slide under oil immersion.

23. Vegetative cells are colorless , endospores are red, and the background is black. Examples of Endospore Staining Positive Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus cereus ,  Desulfotomaculum   spp ,  Sporolactobacillus   spp ,  Sporosarcina   spp , etc. Negative E. coli, Salmonella   spp , etc.

24. Acid-fast stain Purpose The acid-fast stain is performed on samples to demonstrate the characteristic of acid fastness in certain bacteria and the cysts of Cryptosporidium and Isospora . Clinically, the most important application is to detect Mycobacterium tuberculosis in sputum samples to confirm or rule out a diagnosis of tuberculosis in patients. Acid-fast staining was originally pioneered by a scientist named  Paul Ehrlich  in the year 1882. Later, it was modified by  Ziehl  and  Neelson  in 1883. Thus, acid-fast staining is also called Ziehl Neelson staining. It is a type of  differential staining  method used to distinguish between acid-fast and non-acid-fast bacteria. Mycobacterium is an acid-fast bacterium which retains the colour of carbol fuschin even after treatment with a decolourizer . The mycobacteria species retain the primary stain’s colour because they contain mycolic acid in their cell wall.

25. Requirements : Clean glass slide slide holder bacterial suspension inoculating loop Bunsen burner distilled water water bath Ziehl Neelsen stain A cid alcohol Loeffler’s methylene blue oil immersion and microscope.

26. Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia. Preparation of microscope slide: Clean slide with a Kimwipe and alcohol to remove any fingerprints. Draw two circles with your Sharpie on the  bottom  of the slide. Using your inoculation loop, put two  small  drops of water in each circle. Using aseptic technique, remove a  very small  amount of bacteria from the culture tube. Make sure you flame the tube before and after you enter. Smear the bacteria in the drop of water on your slide. You may go out of the perimeter of your circles! Let the slide  air dry completely . Heat-fix the slide by running it through the flame 3-4 times with the ‘smear’ side up. Do not flame the side with the bacteria! Let the slide cool completely and you are ready to stain it.

27. Staining procedure: Cover the smears with a piece of paper towel within the border of the slide. Then, add a drop of distilled water to the centre of the glass slide. After doing this, thoroughly mix the inoculum by a sterilized inoculating loop. Then, heat fix and air dry the prepared smear. Flood the smear with the primary stain ( Carbol fuschin ) and allow it to stand for one minute. Then, flood the smear with the decolourizer (3% HCl) and wash the glass slide with distilled water. Finally, flood the smear with the counterstain (Loeffler’s methylene blue) and allow it to stand for one minute. Again rinse the slide in water. Air-dry the glass slide. Observe the glass slide under oil immersion at 100X objective.

28. Interpretation of Result Examples  of  acid-fast bacteria :  Mycobacterium tuberculosis ,  M. leprae ,  M. smegmatis ,  M. phlei  etc. Examples  of  non-acid fast bacteria :  Escherichia coli ,  Staphylococcus aureus  etc. Acid-fast cells : They appear red coloured , rod shaped and can occur singly or in small groups. Non-acid fast cells : They appear blue coloured .

29. Bacterial Growth Bacteria are unicellular organisms that tend to reproduce asexually by the means of binary fission. Bacterial growth is the increase in the number of bacterial cells rather than the increase in their cell size. The growth of these bacterial cells takes place in an exponential manner, i.e., one cell divides into 2, then 4, then 8, 16, 32 and so on. The time taken for a bacterial cell to double is called generation time. The generation time varies among different species of bacteria based on the environmental conditions they grow in.  Clostridium perfringens  is the fastest growing bacteria that has a generation time of 10 minutes while  Escherichia coli  has a doubling time of 20 minutes.  Mycobacterium tuberculosis  is one of the slowest growing bacteria, taking about 12 to 16 hours to double.

30. Growth Curve In a closed system with enough nutrients, a bacteria shows a predictable growth pattern that is the bacterial growth curve. It consists of four different phases. Phases of the Bacterial Growth Curve Upon inoculation into a new nutrient medium, the bacteria shows four distinct phases of growth. Let us dive into each of the phases in detail – Lag Phase The bacteria upon introduction into the nutrient medium take some time to adapt to the new environment. In this phase, the bacteria does not reproduce but prepares itself for reproduction. The cells are active metabolically and keep increasing in size. The cells synthesise RNA, growth factors and other molecules required for  cell division .

31. Log Phase Soon after the lag phase, i.e., the preparation phase, the bacterial cells enter the log phase. The log phase is also known as the exponential phase. This phase is marked by the doubling of the bacterial cells. In the exponential phase, the cells begin to actively divide through binary fission and double their numbers. The cell number increases in a logarithmic fashion such that the cell constituent is maintained. The log phase continues until there is depletion of nutrients in the setup. The stage also comes to a stop if toxic substances start to accumulate, resulting in a slower growth rate. The cells are the healthiest at this stage and researchers prefer to use bacteria from this stage for their experimental processes. Plotting this phase on the bacterial growth curve gives a straight line. Stationary Phase In the stationary phase, the rate of growth of the cells becomes equal to its rate of death. The rate of growth of the bacterial cells is limited by the accumulation of toxic compounds and also depletion of nutrients in the media. The cell population remains constant at this stage. Plotting this phase on the graph gives a smooth horizontal linear line. Death Phase This is the last phase of the bacterial growth. At this stage, the rate of death is greater than the rate of formation of new cells. Lack of nutrients, physical conditions or other injuries to the cell leads to death of the cells.

32. Two types of asexual reproduction for bacterial cells’ growth are: Budding:  Here, the bacterial cell protrudes in the form of a bud — a bulb-like projection — at a particular site due to cell division. Binary Fission:  The bacterial cells divide into two daughter cells in this process. During binary fission, bacterial cells replicate their genetic material, divide their cellular content into the divided cells, and transfer one each into two daughter cells produced through fission.

33. Nutrient and Physical Requirements for Growth It’s essential to provide an optimum environment and nutrient conditions for microbial growth under lab conditions. Chemical or Nutrient Requirement Carbon source:  Carbon makes 50% of the cell’s dry weight. It’s the structural backbone of all organic compounds, and different bacterial species have different carbon requirements: Chemoheterotrophs: These organisms obtain carbon from proteins, lipids, and carbohydrates energy sources. Sometimes, they also get it from complex compounds such as vitamins and growth factors. Chemoautotrophs and Photoautotrophs: They primarily obtain carbon from carbon dioxide (CO 2 ). Nitrogen source:  It’s one of the essential elements required by organisms. It’s involved in building amino acids, RNA, and DNA. Though most species use proteins as their nitrogen source, nitrogen-fixing bacteria obtain nitrogen directly from the atmosphere, while some bacterial species use nitrate salts as their source of nitrogen. Sulfur source:  It is required to form certain proteins and vitamins, such as biotin and thiamine. Bacterial species obtain sulfur from proteins, hydrogen sulfide, and sulfates.

34. Phosphorus source:  It’s required for building DNA, RNA, ATP, and phospholipids. The sources of phosphorus for bacterial species are inorganic phosphate salts and buffers. Trace elements:  These elements are used as cofactors of enzymes; they include iron, copper, zinc, and molybdenum. Oxygen:  Bacteria are classified into five groups based on their oxygen requirements [3] : Obligate aerobes: They require oxygen to live; for example,  Pseudomonas Obligate anaerobes: They don’t require oxygen and can’t even tolerate their presence, e.g.,  Clostridium . Facultative anaerobes: These require oxygen but can also live without it; examples are  Staphylococcus  and  coli . Aerotolerant anaerobes: Bacterial species in this group do not require oxygen to live but can tolerate their presence. They can also break down toxic forms of oxygen, e.g.,  Lactobacillus caries. Microaerophiles : They require only a small concentration of oxygen to live. These microbes are sensitive to toxic forms of oxygen like hydroxyl radicals. Culture Medium:  In lab conditions, a nutrient medium containing nutrients and minerals formulated according to the needs of the bacterial species is required. Moreover, according to the nature of bacterial species,  culture media can be prepared  as solid, semi-solid, or liquid by varying agar concentrations

35. Physical Requirements / Physical factors affecting : pH:  Many bacterial species prefer a pH of 6.5-7.5. Based on the preferred pH for their growth, the bacterial species are categorized into three groups Acidophiles: They are acid-loving and prefer to grow at a pH between 0.1 to 5.4. Neutrophiles: They prefer habitats with a pH level of 5.4 to 8.5. Alkaliphiles: They prefer a basic pH level, anything between 7 to 12 or even higher. Temperature:  Different bacterial species survive at different temperatures, and based on their temperature preferences, they are categorized into three groups . Mesophiles: They live at temperatures between 25  o C to 40  o Psychrophiles: They are cold-loving and live at temperatures between 0  o C to 20  o Thermophiles: They live in hot places that are 50  o C to 60  o C in temperature. Osmotic Pressure:  Bacterial cells possess 80-90% water. An increase or decrease in the water content leads to plasmolysis (hypertonic solution) or cell lysis (hypotonic solution).

36. What is Culture Media? Culture media is a gel or liquid that contains nutrients and is used to grow bacteria or microorganisms. They are also termed growth media. Different cell types are grown in various types of medium. Nutrient broths and agar plates are the most typical growth media for microorganisms. Some microorganisms or bacteria need special media for their growth. Culture media or growth media are solid, liquid or semi-solid substances that aid in the proliferation of bacterial populations

37. Bactericidal agents Phenol: Phenol (carbolic acid) is one amongst the oldest antibacterial agents. It acts as a bacteriostat by inhibiting biological process of bacteria at concentrations of 0.1%-1% and is fungicidal in action at concentration of 1%–2%. 5% concentration kills anthrax spores in 48 hours. The antiseptic activity is increased by EDTA and heat temperatures; it's decreased by alkaline medium (through ionization), lipids, soaps, and cool temperatures. Alcohol: Alcohols possess many features desirable for a disinfectant or antiseptic. They have excellent bactericidal efficacy as well as bacteriostatic action as a preservative, some virucidal efficacy (especially against enveloped viruses), and fungicidal efficacy. Alcohols are known for their effectiveness against a wide variety of bacteria and viruses at concentrations of 70%-95%. Alcohols have little killing effect when it comes to spores. Ethyl alcohol, at concentrations of 60%–80%, is a potent viricidal agent inactivating all the lipophilic viruses (e.g., herpes, vaccinia, and influenza virus) and many hydrophilic viruses (e.g., adenovirus, enterovirus, rhinovirus, and rotaviruses, but not hepatitis A virus (HAV) or poliovirus). Isopropyl alcohol is not active against the nonlipid enteroviruses, but is fully active against the lipid viruses. Studies also have demonstrated the ability of ethyl and isopropyl alcohol to inactivate the hepatitis B virus(HBV) and the herpes virus, and ethyl alcohol to inactivate human immunodeficiency virus (HIV), rotavirus, echovirus, and astrovirus

38. Sterilization In microbiology, S terilization can be defined as the complete removal of all forms of microorganisms, both vegetative and spore forms, from a surface or an object. Sterilization is carried out by various physical and chemical methods such that it eliminates around 10 6  log colony-forming units. Sterilization  (or sterilisation ) refers to any process that removes, kills, or deactivates all forms of  life  (particularly  microorganisms  such as  fungi ,  bacteria ,  spores , and  unicellular   eukaryotic  organisms) and other  biological agents  such as  prions  present in or on a specific surface, object, or fluid. Sterilization can be achieved through various means, including  heat ,  chemicals ,  irradiation ,  high pressure , and  filtration . Sterilization is distinct from  disinfection , sanitization, and  pasteurization , in that those methods reduce rather than eliminate all forms of life and biological agents present. After sterilization, an object is referred to as being sterile or  aseptic .

39. Classification of Sterilization Sterilization is achieved by different physical and chemical methods in  microbiology . Sterilization is classified into 2 types – physical sterilization and chemical sterilization. Let us discuss them in detail. Physical Methods of Sterilization Physical sterilization includes the following methods: Heat Sterilization Heat sterilization is the most effective method of sterilization, where the elimination of microbes is achieved by the destruction of cell constituents and enzymes. It is done by two methods: Moist Heat Sterilization:  It is one of the best methods of sterilization. Moist heat sterilization is done with the help of an instrument called an autoclave. An autoclave works on the principle of producing steam under pressure. Thus moist heat sterilization is also known as steam sterilization. The water is boiled in an autoclave at 121-134℃ at a pressure of 15psi. This leads to coagulation of proteins in the microorganism, and they are effectively killed.

40. Physical Methods of Sterilization 2. Dry Heat Sterilization:  This method is used on objects that are sensitive to moisture. Moisture-free heat or dry heat is applied on the surface or objects such that there is denaturation and lysis of proteins which leads to oxidative damage, and ultimately the microbial cell dies out or may even burn. Some methods of dry heat sterilization include incinerators, hot air ovens.

41. Physical Methods of Sterilization Filtration This is a mechanical method of sterilization in microbiology. This method uses membranous filters with small pores to filter out the liquid so that all the bigger particles and microbes cannot pass through. The three steps of filtration are sieving, adsorption and trapping.

42. Physical Methods of Sterilization Irradiation Irradiation is the process of exposing surfaces or objects to different kinds of radiation for sterilization. It is of two types: Non- ionising Radiation:  Ultraviolet radiation is exposed to the object, which is absorbed by nucleic acids of the microorganisms. This leads to the formation of pyrimidine dimers in the DNA strand, which causes the replicative error, and eventually, the microbe dies. Ionising Radiation:  Upon exposure to ionising radiations such as gamma rays and X-rays, reactive oxygen species such as hydrogen peroxide and superoxide ions are formed that oxidise the cellular components of the microbe, and they die.

43. Chemical Methods of Sterilization Chemical methods of sterilization are used in microbiology for biological specimens and plastic equipment. In this method, several chemicals work as bactericidal agents. They can be of two types: gaseous or liquid. Gaseous Sterilization Gaseous sterilization is the method where the object is exposed to gas in a closed, heated and pressurized chamber. The gaseous chemical agents used for sterilization include ethylene oxide, formaldehyde, nitrogen dioxide and ozone.

44. Chemical Methods of Sterilization Liquid Sterilization Liquid sterilization is the process of immersing the object in a liquid such that it kills all the viable microorganisms and their spores. This method is less effective than gaseous sterilization and is used to remove low levels of contamination. Common liquid chemical agents that are used for sterilization include hydrogen peroxide, glutaraldehyde and hypochlorite solution. Cold Sterilization Definition –  It is a process in which sterilization is carried out at low temperatures with the help of chemicals, filters, radiation and all other means excluding high temperatures.  It is done for products that contain heat-sensitive ingredients and yet require sterilization.