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• Many microbial may havd Undesirable consequences

• It is Important to kill or inhibit microorganisms to minimize destructive effects

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• Sterilization defined

  • Process of destroying or removing all living cells, spores, and acellular entities

  • From latin word sterilis which. Eans unable to produce offspring or barren

• Sterilant - chemical agent for sterilization

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• Types of Autoclaves: Pressure Cooker, Common Laboratory, Vertical, Horizontal, Hospital

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• Disinfection defined

  • Killing, inhibition, or removal of disease-causing microorganisms

• Disinfectants - chemical agents for disinfection on inanimate objects

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• Sanitization defined

  • Reducing microbial population to safe levels

• In sanitization Inanimate objects are cleaned and partially disinfected

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• Antisepsis defined

  • Control of microorganisms on living tissues to prevent infection

  • From greek word anti meaning against and sepsis meaning putrefaction

• Antiseptics - chemicals to prevent infection on tissues, they arenot as toxic as disinfectants

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• Antimicrobial Chemotherapy

  • Use of chemical agents to kill or inhibit microorganisms within host tissue

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• Germicide vs Bacteriostatic

  • Germicide kills pathogens but not endospores

  • Bacteriostatic prevents growth

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• Physical Methods of Microbial Control

  • Moist Heat is a method to kill bacteria, viruses, and fungi by degrading nucleic acid abd disrupting cell membranes

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• Steam sterilization

  • Type of moist hear that uses tempersture above 100°C

  • It destroys al lvegetativr cells and endospores

• Autoclave at 121°C and 15 psi

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• Pasteurization

  • Controlled heating below boiling point for preservation and brief heating at 55°C - 60°C

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• Dry hear is the sterilization in the absence of water for example incinerator and oven

• Dry heat oxidizes cell constituents and denature proteins

• Low temperature is a method to inhibit microbial growth and reproduction Through freezing and refrigeration

• -20°C stops microbial growth

• -30°C or -70°C is good for long term storage

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• Filtration is a method that removes microorganisms rather than directly destroying them

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• High-Efficiency Particulate Air (HEPA) filters

  • Used for air sterilization and removes 99.97% of 0.3 um particles

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• Radiation another physical method of microbial control

  • Ultraviolet (UV) is around 260 nm and is little to microorganism but it does not effectively penetrate glass, dirt films, water, and other substances

  • Ionizing Radiation is an excellent sterilizing agent and it penetrates deep into objects it destroys bacterial endosphere and vegetative cells both in prokaryotic and eukaryotic cells

  • Gamma radiation from a cobalt 60 source is used to sterilize and pasteurize meat and other food

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Characteristics of a Desirable Disinfectant:

• Effective against various infectious agents at low concentrations and in the presence of organic matter

• Relatively inexpensive

• Low surface tension for entering cracks in surfaces

• Soluble in water and lipids for penetration into microorganisms

• Odorless or with a pleasant odor

• Stable upon storage

• Must not be toxic to people or corrosive to common materials

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Phenolics:

• Act by denaturing proteins and disrupting cell membranes

  • Effective in the presence of organic material

  • Remain active on surfaces long after application

  • Can cause skin irritation

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Alcohols:

• Bactericidal and fungicidal but not sporicidal

  • Act by denaturing proteins and possibly dissolving membrane lipids

  • 70% or 80% ethanol ans isopropanol

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Halogens:

• Iodine used as a skin antiseptic and kills by oxidizing cell constituents and iodinating cell proteins

• Chlorine used as a disinfectant for water supplies, oxidizing cellular materials And destructs vegetative bacteria and fungi although not spores

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Heavy Metals:

• Combine with proteins and inactivate them

  • May precipitate cell proteins

  • Examples are silver nitrate solution and copper sulfate

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Quaternary Ammonium Compounds:

• Detergents with antimicrobial activity

  • Effective disinfectants

  • Disrupt microbial membranes and denature proteins

  • They are amphipathic organic cleaning agents

  • Samples include benzalkonium chloride and cetylpyridinium chloride

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• Aldehydes Combine with nucleic acids and proteins to inactivate them by Cross-linking and alkylating molecules

  • Examples include formaldehyde and glutaraldehyde

• Sterilizing gases

  • ethylene oxide - it is microbesidal and sporicidal and kills by combining with cell proteins

  • betapropiolactone - it destroys microbes more readily than ethylene oxide but it does not penetrate materials as well as it is carcinogenic

  • vaporized hydrogen peroxide - toxic and kills wide variety of microbes

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Chemotherapeutic Agents:

• Used internally to kill or inhibit microbe growth within host tissues

  • Selective toxicity

  • Example: antibiotics

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Microbial Culture Medium:

• Mixture supporting growth and differentiation of microorganisms

  • Microorganisms growing in it are the culture

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Components of Culture Media:

• Mimics organism's natural habitat

  • Includes protein, nitrogen, carbohydrates, agar, dyes & indicators, enriching substances

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Agar:

• Developed by Fanny Hesse

  • Extracted from red seaweed

  • Used as a solidifying agent for solid medium

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Types of Culture Media Based on Consistency:

• Solid Media with 1.5% to 2% agar

  • Used for growing microorganisms in full form, preparing pure cultures, and studying colony characteristics

  • Samples are blood agar nutrient agar mcconkey agar and chocolate agar

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B. Semi-solid Media

• Contains 0.2% to 0.5% agar

  • Appears as a soft, jelly-like substance

• Used for studying microorganism motility and cultivating microaerophilic bacteria

• Examples include Hugh and Leifson’s oxidation fermentation medium, Stuart’s and Amies media, and Mannitol motility media

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C. Liquid Media

• No solidifying agent

  • Also known as broths

• Allows large growth of bacterial colonies

• Used for profuse growth of microorganisms and fermentation studies

• Examples include Tryptic soy broth, phenol red carbohydrate broth, MR-VP broth, and nutrient broth

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TYPES OF CULTURE MEDIA BASED ON NUTRITIONAL COMPONENT

A. Simple media

• Supports growth of non-fastidious microbes

• Primarily used for isolating microorganisms

• Examples: nutrient broth, peptone water, nutrient agar

B. Complex media

• Contains nutrients with unknown concentrations

• Added to bring about specific characteristics in microbial strains

• Examples: tryptic soy broth, blood agar, nutrient broth

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C. Synthetic media

• Chemically defined media made from pure chemical substances

• Known concentration of ingredients

• Commonly used in scientific research

• Example: Czapek Dox Medium

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TYPES OF CULTURE MEDIA BASED ON APPLICATION OR CHEMICAL COMPOSITION

A. Basal media

• Simple media with carbon and nitrogen sources

• Boosts growth of various microorganisms

• Considered non-selective media

• Used for isolating microorganisms in labs or sub-culturing processes

• Examples: nutrient broth, nutrient agar, peptone water

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B. Enriched media

• Additional substances like blood, serum, or egg yolk added to basal medium

• Supports growth of fastidious microorganisms

• Examples: Chocolate media, Blood agar

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C. Selective media

• Allows growth of specific microbes while inhibiting others

• Used for isolating microorganisms

• Selective growth determined by adding substances like antibiotics, dyes, bile salts, or adjusting pH

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D. Differential or Indicator media

• Contains indicators like dyes or metabolic substrates

• Differentiates bacterial colonies based on color when utilizing components

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• Examples of differential media and their functions for differentiating microbial species

  • Blood agar it differentiates bacteria based on their hemolysis

    • Alpha hemolysis will show brown color an example for this is a escherichia coli it is only partial hemolysis

    • Beta hemolysis is complete hemolysis an example for this is streptococcus pyogenes it shows that the agar will lose its red color

    • Gamma hemolysis shows no hemolysis for an example for this is staphylococcus epidermidis agar will still appear in red color

  • Mannitol salt agar shows the fermentation of mannitol by staphylococcus aureus which causes the media to change to yellow

    • Coagulase positive staphylococci will show yellow color an example for this is staphylococcus aureus

    • Coagulase negative staphylococci and cause fermentation which is why they appear in pink color an example for this is micrococcus luteus

    • Staphylococcus epidermidis will show a light pink color because it is only partial fermentation

  • MacConkey Agar differentiate the gram negative bacteria based on their lactose metabolism

    • Lactose fermenting bacterial like escherichia coli klebseilla spp citrobacter enterobacter will show a pink red colony in MacConkey Agar

    • Lactose non fermenters like salmonella shigella proteus providentia pseudomonas and morganella will form a pale or colorless colony in MacConkey Agar

  • Thiosulfate citrate bile salt sucrose agar also known as tcbs is used to differentiate organisms that ferment sucrose

    • Bacterias like v.cholerae that ferment sucrose will form a slightly flattened yellow colonies having opaque centers and translucent peripherals in tcbs agar

    • Bacterias like v.haemolyticus that cant ferment sucrose will form a green to blue green colonies

  • Transport media is the media used for clinical specimens which are required to be transferred immediately to maintain viability

  • Anaerobic media is used for anaerobic bacteria

  • Assay media is used for amino acid vitamins and antibiotic assay

    • Antibiotic assay media is used to determine the antibiotic potency of microorganisms

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• Pure Culture vs. Mixed Culture

  • Pure Culture: Contains only one species of microorganisms

  • Mixed Culture: Contains two or more organisms

  • Obtaining pure cultures requires starting with a sterile growth medium and keeping unwanted microorganisms out

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• Aseptic Technique

  • Involves target-specific practices to reduce contamination

  • Proper use significantly reduces the risk of contamination

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• General Principles of Aseptic Technique

  • Culture medium and container must be sterilized

  • Container must be covered

  • Instruments and fluids touching the medium must be sterilized

  • Work area should not be contaminated

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• Inoculation

  • Introducing microorganisms into a culture medium

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• Inoculating loop and needle

  • Devices for transferring microorganisms into a culture medium

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• Aseptic Technique in Inoculation of Bacteria

  • Steps like flaming loop and tube, transferring, capping, and putting back

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• solid media

  • Different forms include agar slants, deep tubes, and plates

• Types of inoculation in plated media

  • Continues streak

  • Interrupted streak

  • Multiple interrupted streak

  • Multiple inoculation

  • Radial streak

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• Colony Morphology and Growth Patterns

  • Different species produce different-looking colonies

  • Observing colony morphology helps identify microorganisms

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• Colony Morphology

  • Visual characteristics of bacterial colonies on agar plates

  • Important for identifying microorganisms in the lab

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• Characteristics of a Colony

  • Includes size, form, elevation, margin, surface, opacity, and color

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• Elevation of a colony

  • Described as raised, convex, flat, umbonate, or crateriform

  • Side view of colony

• Size of colony

  • Dismeter of the colony

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• Surface of a colony

  • Appearance of the surface of colony

  • Can be smooth, glistening, rough, wrinkled, or dull

• Opacity

  • Can be transparent, opaque, translucent, or iridescent

• Color is the pigmentation of colony

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• Growth Patterns in Agar Slant

  • Can be filiform, echinulate, beaded, effuse, arborescent, rhizoid

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• Growth Patterns in Liquid Media

  • Can be seen as clear, turbid, flocculent, pellicle, sediment, and no growth

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• Staining Techniques

  • Importance of staining to visualize microorganisms under the microscope

  • Different stains adhere to cell structures, creating contrast for visibility

  • Stain is a substance that adheres to a cell and gives color

  • Stain have different affinities to different structures thereby creating contrast and make the structures more visible

  • Microbes must be fixed and stain correctly to increase visibility accentuate specific features and preserve them for future

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• Preparation of Bacterial Smear

  • Apply bacterial cells on a slide

  • Spread using an inoculating loop

  • Fix cells to the slide using heat or chemicals

    • Heat fixation

    • Methanol fixation

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• Simple/Direct Staining

  • Uses a single dye to color the microbe

  • Positive Staining

    • Dye stains molecules within the microbe

  • Negative Staining

    • Dye stains the background around the microbe

• Examples of dyes

  • Methylene blue, crystal violet (Positive Staining)

  • Nigrosin (Negative Staining)

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• Differential Staining

  • Uses multiple dyes to differentiate cell types

  • Primary dye colors all cells

  • Decolorizing agent removes dye from some cells

  • Secondary stain also known as counterstain provides color to decolorized cells

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• Gram Staining

  • Differentiates bacteria based on cell wall structure

  • Gram-positive bacteria stain purple because they have thick peptidoglycan layer

  • Gram-negative bacteria stain red because they have thin peptidoglycan layer

• Acid fast staining

  • A differential stain that distinguishes bacteria that contains a wax like lipid called mycolic acid

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• Special Staining

  • Used to stain bacterial structures for identification

• Endospore Staining

  • Distinguishes endospores in bacteria

  • Heat used to drive malachite green into endospore

  • Uses malachite green as primary stain

  • Uses safranin as counterstain

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• Capsule Staining

  • Identifies bacteria with a capsule around cell walls

  • Capsules enhance disease-causing ability

  • A negatively charged capsule will repel the dye producing a clear zone surrounding the bacteria

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• Flagella Staining

  • Identifies motile bacteria with flagella

  • Flagella visualized after coating with dye

  • More than is used to fix the stain on the flagella since flagella are two thin and can't be seen with a light microscope

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• Biochemical Characterization of Microorganisms

  • Microscopic observation provides limited information

  • Biochemical reactions aid in identifying bacteria

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• IMViC Tests

  • Distinguish between Enterobacteriaceae based on metabolic byproducts

  • Used for characterizing Actinobacteria

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• Indole Test

  • Screens for the ability to produce indole from tryptophan

  • Red color indicates a positive test

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• Methyl Red Test

  • Detects acid production during glucose fermentation

  • Culture medium turns red at pH 4.4 or below

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• Voges-Proskauer Test

  • Determines acetoin production from glucose fermentation

  • Acetoin oxidized to diacetyl, forming a pinkish-red product

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• Voges-Proskauer Test Procedure

  • Prepare a culture in MR-VP broth

  • Add a-naphthol and KOH to the broth culture

  • Shake the tube to expose the medium to oxygen

  • Allow the tube to stand for at least 30 minutes

  • Read the test result within 1 hour

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• Citrate Test

  • Tests organism's ability to use citrate as an energy source

  • Bacteria metabolize citrate, breaking down ammonium salts to increase alkalinity

  • pH shift turns bromthymol blue from green to blue above pH 7.6

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• Citrate Test Procedure

  • Inoculate sample on Simon Citrate agar slants

  • Incubate at 35°C for 18 to 48 hours

  • Intense Prussian blue growth indicates a positive test