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MODULE 1 Archaea (environment) LESSON 1: MICROBIOLOGY EUKARYOTES PROKA EUKA Fungi NUCLEUS Absent...

MODULE 1 Archaea (environment) LESSON 1: MICROBIOLOGY EUKARYOTES PROKA EUKA Fungi NUCLEUS Absent Present NOT CELL Virus NUCLEAR Absent Present MEMBRANE DIVISIONS OF MICROBIOLOGY Organisms studied CELL WALL Peptidoglycan Cellulose/Chitin 1. Bacteriology - bacteria LYSOSOMES Absent Present 2. Protozoology - protozoa MITOCHOND Absent Present 3. Mycology - fungi RIA 4. Virology - virus RIBOSOMES Smaller/70s/ Bigger/80s/FR 5. Parasitology - parasites FR only and AR 6. Phycology - algae ER Absent Present Health related 1. Etiology – identification of causative DNA Circular / Linear / dsDNA dsDNA agent of disease 2. Epidemiology – study of spread , of REP Bacteria Fungi / ORGANISMS Protozoa disease 3. Immunology – study of immune FR - FREE AND SCATTERED RIBOSOMES system AR - ATTACHED RIBOSOMES 4. Chemotherapy – treatment of disease dsDNA - DOUBLE STRANDED DNA with chemical compounds Microbiology is the study of 5. Infection control – control of spread organisms too small to be seen of infectious disease with the nakedeye. Processes, Functions Microorganisms 1. Microbial metabolism Microscope 2. Microbial genetics 3. Microbial ecology MICROORGANISMS 1. Bacteria - simple, single cell. IMPORTANCE OF MICROBIOLOGY / 2. Fungi - single & multi cell forms MICROORGANISMS - yeast, filamentous molds, and A. Causative agents of infectious complex fungi. diseases 3. Protists - single cell, some multicellular B. Normal flora - good bacteria in the - algae, protozoans, slime molds. body 4. Viruses - acellular, intracellular 1. Beneficial metabolic functions parasites. 2. Antagonistic effect - prevents 5. Worms - multicellular, more complex. invasion pathogens, overgrowth of potential pathogens TYPES OF CELL C. Environmental importance PROKARYOTES 1. Decomposer Eubacteria : Bacteria Mycoplasma, 2. Produce oxygen Chlamydia, Spirochetes, Rickettsia, 3. Food chain Actinomycetes 4. Sewage treatment  D. Industrial importance JOHN NEEDHAM (1749) 1. Food industry 1. Performed experiments similar to 2. Brewing industry Redi’s 3. Pharmaceutical industry 2. Introduced the first culture medium for 4. Genetic engineering microbial growth. E. Research - genetics, metabolism 3. Utilized infusion broth prepared by 1. Simple cell structure boiling meat, grain, etc. to extract 2. Rapid rate of growth nutrients. 3. Inexpensive to culture 4. Turbidity indicated growth. 5. Broth put in flasks, some were sealed LESSON 2: DEVELOPMENT OF with corks, some were not. 6. Results were inconsistent – all flasks EARLY MICROBIOLOGY became cloudy. THEORY OF SPONTANEOUS GENERATION 7. Reasons: organisms in air or flasks, 1. This theory existed to explain the improper seal. origin of some forms of life. 2. Living organisms arose LAZZARO SPALLANZANI (1776) “spontaneously” from nonliving, 1. Repeated Needham’s experiments. decaying organic matter. 2. Boiled broth after placing in flasks. 3. Believed to explained origin of 3. Sealed flasks by melting necks. “animalcule” 4. Results are more consistent with Redi’s. 5. Occasionally sealed flask cloudy. Biogenesis - Life came from living things 6. Not accepted because heating destroyed, degraded “vital force”. Abiogenesis (Spontaneous Generation) - Life came from non-living things unless vital LOUIS PASTEUR (1861) force which is heat. Hypothesis was designed (FATHER OF MODERN IMMUNOLOGY) by Aristotle. 1. Performed experiments to disprove Theory of SG. FRANCESCO REDI a. Filtered air through a cotton (FATHER OF PARASITOLOGY) plug. Placed plug in infusion 1. Performed experiments that disproved broth,broth became cloudy - theory of SG for more complex forms organisms present in the air. of life (began approx.1668). b. Placed boiled infusion broths in 2. Utilized jars containing meat. Some “swan-necked” flasks were covered, some were not. c. Flasks remained sterile unless 3. Maggots appeared in uncovered jars. tilted or neck broken. 4. Results not accepted for microscopic 2. His experiments were accepted as organisms. disproof of the theory of Spontaneous 5. Introduced experimental procedure for Generation. disprove Spontaneous Generation. 3. Additional work: 6. Spontaneous Generation took another a. Pasteurization - to prevent 200 years to disprove. spoilage of wine. b. Introduced “Germ Theory of Disease” after discovering  silkworm disease caused by MARTINUS BEIJERINCK (1884 - 85) protozoans. a. Discovered filterable agents called c. Developed Pasteur treatment for “viruses” (toxins, poisons). preventing rabies using dried b. Infectious agents in tobacco plant spinal cord from infected dogs. fluids. c. Assumed soluble toxin in filtrate GOLDEN AGE OF MICROBIOLOGY caused disease (APPROX. 1875 - 1918) d. Called “viruses” (Latin for toxins, 1. Period (about 50 years) of rapid poisons) development. 2. Causes of diseases identified, control PAUL EHRLICH (1910) methods developed, work began on a. Introduced the concept of viruses. chemotherapy. 3. Robert Koch b. Use of salvarsan for the treatment of syphilis. ROBERT KOCH While studying anthrax ( a disease of cattle) : ALEXANDER FLEMING (1928) a. Identified a bacterium as the cause of a. Discovered the first antibiotic - anthrax (first bacterium that caused a penicillin. disease). b. Produced by mold that contaminated b. Introduced “solid medium” using agar. bacterial culture plates. Observed differences in colony c. Led to discovery of other fungi that morphology. secreted antibacterial substances Introduced the inoculating loop to (antibiotics). transfer bacteria and prepare pure cultures. c. Introduced “Koch’s Postulates” and the MODULE 2 concept that a disease is caused by a LESSON 1: BACTERIA single organism. THE GERM THEORY OF DISEASE Microbes (germs) cause disease and specific microbes cause specific diseases in the late 1870's. Anthrax - disease of cattle/sheep; also in humans Particular microbes cause particular diseases. BACTERIAL CELL SHAPE/ARRANGEMENT EDWARD JENNER (1796) Cocci/Coccus - Singles, pairs, irregular 1. Developed smallpox vaccination. clusters, tetrads, cubical packets of 8 2. Used fluids from cowpox (vaccinia) Bacilli/Bacillus - Singles, pairs, chains, lesions. parallel packets. JOSEPH LISTER (1865) SPIRAL A. Introduced the “antiseptic technique”. 1. Vibrios - single curve, comma shaped B. Use of phenol as disinfectant.  2. Spirillum - rigid, two or more curves, corkscrew shape 3. Spirochetes - flexible, two or more curves, wavy - move by flexing bodies. ACID FAST STAINING It is a type of differential staining method used to distinguish between the acid-fast and non-acid fast bacteria. CLASSIFICATION BASED ON FLAGELLA Motility - movement Acid-fast organisms have a lipoid capsule Motile/Non motile that has a high molecular weight and is waxy Arrangement basis for classification at room temperature. This makes the Monotrichous: 1 flagella organism impenetrable by aqueous-based Lophotrichous: tuft at one end staining solutions. The lipoid capsule of an Amphitrichous: both ends acid-fast organism stains with carbol-fuchsin Peritrichous: all around bacteria and resists decolorization with dilute acid rinse. RULES BACTERIAL CLASSIFICATION BASED ON 1. All cocci are gram positive except STAINING REACTIONS Neisseria, Veillonella, and Moraxella. 2. All bacilli are gram negative except GRAM STAIN Bacillus, Mycobacterium Clostridium The organism that retains the primary stain Corynebacterium, Listeria, in the Gram stain procedure and appears Actinomycetes, Lactobacillus, purple-brown under the microscope are Nocardia, Arcanobacterium termed Gram-positive organisms. Gardnerella 3. All bacteria are non-acid fast except Whereas the organisms termed Mycobacterium and Nocardia. gram-negative do not take up the primary stain and appear red under the microscope. LESSON 1.2: DIFFERENTIAL THE PROPER SEQUENCE OF REAGENTS IN THE GRAM STAIN PROCEDURE STAINING 1. The crystal violet stain as the primary GRAM STAIN stain Gram staining results are used to 2. Then the mordant (gram iodine) differentiate bacteria as gram-positive 3. The decolorizer or gram-negative. 4. Lastly, the counterstain. The The staining technique was named counterstain for the gram stain after the Danish bacteriologist, Hans procedure can be safranin or basic Christian Gram. fuchsin solution. Hans Gram, the danish bacteriologist was the first to introduce it in 1882, for CRYSTAL VIOLET STAINING REAGENT the identification of Components of Solution A for crystal violet pneumonia-causing organisms. staining reagent The organism that retains the primary 2g of Crystal violet (certified 90% dye stain in the Gram stain procedure and content) appears purple-brown under the 20ml of Ethanol, 95% (vol/vol) microscope are termed Gram-positive organisms. Components of Solution B for crystal violet Whereas the organisms termed staining reagent gram-negative do not take up the 0.8 g of Ammonium oxalate primary stain and appear red under 80ml of Distilled water the microscope. About 90% of the gram-positive GRAM’S IODINE bacteria cell wall is made of thick Components layers of peptidoglycan whereas the gram-negative bacteria cell wall is 1.0 g of Iodine composed of high lipid content as well 2.0g of Potassium iodide as thin layers of peptidoglycan that 300 ml of Distilled water only make up 10% of the cell wall. Formation Equipment Grind the potassium iodide and iodine Bunsen burner together. With continuous grinding add Alcohol-cleaned microscope slide water slowly until the iodine is Slide rack dissolved. Then store in amber bottles. Microscope DECOLORIZING AGENT Gram stain reagents This is made up of: Crystal violet as the primary stain 50 ml Acetone Gram’s iodine solution as the mordant 50 ml Ethanol (95%) Acetone or ethanol as the decolorizer Safranin or 0.1% basic fuchsin solution COUNTERSTAIN – SAFRANIN as the counterstain Stock solution Water The components of the stock solution are: 2.5g Safranin O 100 ml 95% Ethanol Working solution solvent treatment. Based on the cell To form the working solution, combine wall structure of bacterial cells, the 10 ml of the Stock Solution with 90 ml gram-positive microbes have higher of distilled water. peptidoglycan content, while the gram-negative microbes have higher STEPS OF A GRAM STAIN PROCEDURE lipid content. Application of the primary stain (crystal violet) Counterstaining The first of the gram stain steps is the The fourth and final phase of the gram application of the crystal violet stain steps is to use a counterstain. (primary stain) to a heat-fixed smear. Usually, the counterstain used in the In this step, the crystal violet dye is gram staining procedure is Safranin or used for the slide's initial staining. basic fuchsin stain. This process is known as counterstaining and the aim Addition of a mordant (Gram's Iodine) is to give the decolorized The second step in gram staining is the gram-negative bacteria pink color for addition of gram's iodine which is also easier identification. known as fixing the dye. Iodine serves as a mordant in the gram staining APPLICATIONS OF GRAM STAINING procedure. This involves using iodine to Used in research to classify the form a crystal violet-iodine (CV-Iodine) bacteria into Gram-positive and complex to prevent easy removal of Gram-negative the crystal dye. Therefore, the role of Used in diagnostic labs for gram iodine in the gram stain identification of the pathogen procedure is to fix the primary stain Used in hospital for choosing spectrum and prevent the crystal violet from of antibiotic for treatment before leaving the cell. complete identification of bacteria Used to study the morphology of Rapid decolorization bacteria Decolorization with alcohol, acetone, or a mixture of alcohol and acetone is ACID FAST STAINING the third phase of the steps in gram Acid-fast staining was originally pioneered staining. This third step of by a scientist named Paul Ehrlich in the year decolorization is a very crucial step 1882. among the 4 steps of a gram stain. This is because prolonged exposure to a Later, it was modified by Ziehl and Neelson in decolorizing agent can remove all the 1883. stains from both gram-positive and gram-negative bacteria. Thus, acid-fast staining is also called Ziehl Solvents of ethanol and acetone are Neelson staining. often used as decolorizers to remove the dye. The basic mechanism behind It is a type of differential staining method this particular gram stain process is used to distinguish between the acid-fast the ability of the bacterial cell wall to and non-acid fast bacteria. retain the crystal violet dye during REQUIREMENTS OF ACID-FAST STAIN Ziehl Neelson Carbol fuchsin Acid alcohol Loeffler’s Methylene blue LESSON 2: FACTORS AFFECTING BACTERIAL GROWTH REQUIREMENTS FOR BACTERIAL OXYGEN GROWTH 1. Bacteria require oxygen for energy 1. Temperature production 2. Oxygen 2. Bacteria can be divided into four 3. pH groups based on requirement of 4. Moisture gaseous oxygen: 5. Light a. Aerobes - require the presence 6. Nutrients of gaseous (molecular) oxygen b. Anaerobes - require absence of TEMPERATURE gaseous oxygen (utilize O in 1. All bacteria grow within temp. range; oxygen containing compounds - minimum, maximum chemical oxygen). 2. Optimum - temp. of most rapid c. Facultative - either condition reproduction (Facultative anaerobes, 3. Starting at minimum temp. growth facultative aerobes) slowly increases d. Microaerophilic - require the Reaches maximum rate at optimum. presence of small amounts of As temp. continues to rise, growth oxygen (2% - 10%) - reduced rapidly decreases. oxygen tension Bacteria divided into three groups according pH to growth temperature : 1. Concentration of H+, OH- ions a. Mesophiles - 20℃ - 45℃(50℃) 1 ----------------------7 ----------------------14 Ex. - environmental bacteria, 2. Bacteria grow within a pH range pathogen 3. Optimum pH - pH at which maximum b. Thermophiles - 45℃(50) - 80℃ growth occurs Extreme thermophiles (hyperthermophiles) - up to 110℃ Divided into three groups according to pH Ex. - hot springs, volcanoes in ocean, range : decomposers a. Neutrophiles - c. Psychrophiles - 0℃ - 20℃Ex. - cold b. Acidophiles - 0 to 6 springs, lakes; polar regions; 1.) Organisms used in fermentation refrigerator. reactions ® acid (i.e. vinegar, dairy products) 2.) Ex. pathogen: Helicobacter pylori - stomach ulcers c. Alkalinophiles - 8 to 12 Ex : Soil bacteria  d. Optimum pH 7.0 - 7.2 7.35 to 7.45 Based on their energy source bacteria can be grouped into 4 major types – MOISTURE (WATER), OSMOTIC PRESSURE Photosynthetic Bacteria: All metabolically active bacteria require 1) Photoautotrophs and presence of water 2) Photoheterotrophs – Chemosynthetic a. Cells largely water Bacteria: b. Most nutrients, wastes soluble in water 3) Chemoautotrophs and to cross cell membranes. 4) Chemoheterotrophs c. Site of metabolic reactions (cytoplasm) NUTRITIONAL REQUIREMENTS OSMOTIC PRESSURE 1.. Basic requirements for growth : a. Exerted by solutes in water a. Carbon - building blocks of cell b. Increase o.p. outside cell - water components leaves cell b. Nitrogen - production of ( very high o.p. - dehydrates cell ) proteins, nucleic acids c. Decreased o.p. outside cell - water c. Hydrogen - occur in organic enters cell compounds ( very low o.p. - lysis of cell ) d. Oxygen - involved in the d. Halophiles - require the presence of production of energy 3% NaCl e. Minerals, Trace Elements - ( extreme halophiles - 20 to 30% NaCl required in small amounts ) 2. Special metabolites ( growth factors ) a. Substances required for growth SALINITY that the cell cannot produce Halophiles: Bacteria that specifically using the basic requirements require NaCl for growth Moderates already listed. Halophiles: Grow best at 3% NaCl ( Ex. : vitamins, amino acids, solution Many ocean dwelling carbohydrates, blood factors ) bacteria b. Organisms may be described as Extreme Halophiles: Grow well at NaCl being fastidious. concentrations of greater than 15% e.g salt lakes, pickle barrels Two types organisms based on source of nutrients : LIGHT ( RADIATION ) a. Autotrophs - utilize inorganic 1. Very small group photosynthetic compounds bacteria (cyanobacteria) - require UV ( C - CO2, carbonates; N - NH4, N2, light NO3 ) 2. Non Photosynthetic bacteria b. Heterotrophs - utilize organic (eubacteria) - UV light is lethal compounds (causes mutations) ( C - CHO, lipids; N - proteins ) 1) Saprophytes - nonliving organic SOURCE OF ENERGY material Bacteria are found in almost every 2) Parasites - viable (living) environment because they can use organic material widely different energy sources.  To test microbial contamination in any sample. To check antimicrobial agents and preservatives. To observe microbe colony type, its color, shape, cause. To differentiate between different colonies. To create antigens for laboratory use. To estimate viable count. LESSON 3: BACTERIAL CULTURE To test antibiotic sensitivity MEDIA CULTURE MEDIA CLASSIFICATIONS - source of nutrients to support the Classification based on consistency growth of the microorganisms in-vitro. - liquid media - helps in the growth and counting of - semi-solid media microbial cells, selection of - solid media microorganisms, and survival of microorganisms. - can be liquid or gel Common ingredients of culture media Peptone- source of carbon and nitrogen. Beef extract- source of amino acid, vitamins, minerals. Yeast extract- source of vitamin, carbon, nitrogen. Classification based on nutritional Distilled water component Agar- solidifying agent. Basal media - basically simple media that How to prepare culture media? support most non-fastidious 1. Sterilize bacteria. 2. Weight - Peptone water, nutrient broth 3. Dissolve and nutrient agar considered 4. Sterilized basal medium 5. Dispense Enriched media APPLICATION OF CULTURE MEDIA - used to grow nutritionally To culture microbes. exacting (fastidious) bacteria. To identify the cause of infection. - Addition of extra nutrients in the To identify characteristics of form of blood, serum, egg yolk microorganisms. etc, to basal medium makes To isolate pure culture. them enriched media. To store the culture stock. To observe biochemical reactions.  - Blood agar, chocolate agar, indicators (such as neutral red, Loeffler’s serum slope etc are phenol red or methylene blue) few of the enriched media. added to the medium to visibly indicate the defining Blood agar characteristics of a - prepared by adding 5-10% (by microorganism. volume) to a basal medium such as nutrient agar or other blood Mueller Hinton Agar. agar bases. Disc diffusion sensitivity tests for - useful in demonstrating antimicrobial drugs should be carried out on hemolytic properties of certain this media as per WHO recommendation to bacteria. promote reproducibility and comparability of results. Chocolate agar - also known as heated blood agar LESSON 3.2 METHODS OF or lysed blood agar. CULTURING MICROORGANISMS - The procedure is similar to that FIVE BASIC TECHNIQUES of blood agar preparation 1. Inoculate except that the blood is added - Producing a pure culture while the molten blood agar - Introduce bacteria into a growth base is still hot. medium using “aseptic - This lyses the blood cells and technique” to prevent releases their contents into the contamination. medium. This process turns the - Tools: Bunsen burner, loop. medium brown, hence the name. Needle, etc Selective media - designed to inhibit unwanted commensal or contaminating bacteria - help to recover pathogens from a mixture of bacteria. - While selective media are agar based, enrichment media are liquid in consistency. 2. Incubate - growing microbes under proper Differential media or indicator conditions media - Allow organisms to grow under - distinguish one microorganism the optimal conditions type from another growing on - Temperature, with or without the same media. oxygen etc - uses the biochemical 3. Isolation characteristics of a - Colony on media, one kind of microorganism growing in the microbe, pure culture: isolation presence of specific nutrients or  on general and special - treatment to destroy all microbial life (even “differential media” destroying bacterial endospores and fungal - General growth media: NA, TSA spores); there are no degrees of sterility! - Differential: McConkey, EMB, SS - These have dyes, salts, inhibiting DISINFECTION (SANITATION) agents : see differences on - treatment to reduce the number of plates pathogens to a level at which they pose no 4. Inspection danger of disease; disinfectants are used to - Observation of characteristics kill microbes on inanimate objects (most (data) disinfectants are too harsh for use on - Colony Morphology, Microscopic delicate tissue); most disinfectants do not kill examination (gram stain) spores. - Systematic recording of “DATA” 5. Identification ANTISEPSIS - use of data, correlation, to ID - kill microbes or inhibit their growth on skin organism to exact species or other living tissue; Antiseptics are applied - Correlating data from all to living tissue. observations to ID organism to species SANITIZER - Resources: flow charts, Bergey’s – typically used on food-handling equipment manual etc. and eating utensils to reduce bacterial - Ex. Gram – bacilli, ferments numbers so as to meet public health lactose, green sheen on EMB: standards (may mean just washing with soap E.coli in some cases). - Gram + cocci, grape like clusters, golden yellow colonies, catalase “-STATIC” +, coagulase +, resistant to - treatments that inhibit rather than kill; ex. Methicillin (MRSA) refrigeration. (bacteriostatic, fungistatic, - Staphylococcus aureus etc.) STAINING “-CIDAL” Simple stain – one dye - treatments that kill. (bactericidal, Differential stain – complex procedure, fungicidal, viricidal, etc.) (germicidal is a see difference between cells more general) ❖ Grams + and (-) ❖ Acid fast + and (-) CHEMOTHERAPEUTIC AGENTS ❖ Negative – acid dye stains - chemicals, incl. antibiotics, used to treat background and cells are white disease (cell wall repels stain) ❖ Capsule – modified negative Physical Methods Of Microbial Growth stain to show capsule layer Control A. Heat LESSON 4: CONTROL OF 1. Advantages - simple, inexpensive, effective penetrates MICROBIAL GROWTH to kill microbes throughout the Some Important Terms Defined: object; best method if material STERILIZATION  being treated is not damaged by treatment: heat to 63oC heat. for 30 min. or 72oC for 15 2. Mode of Action - denatures sec. proteins. 3. Treatments B. Cold a. Dry Heat Sterilization 1. Effect - microbiostatic; does not - ex. flaming loops, tubes sterilize; slows down enzymes in lab & hot air ovens a. Refrigeration (171oC, 1hr., 160oC for 2 hr., - preserves food because it stops 121oC for 16 hrs.); used to the growth of most species of sterilize materials that can microbes (slows chemical withstand high temps. & reactions); most disease-causing any materials damaged by microbes are mesophiles, not moisture. psychrophiles; an exception is Listeria spp., which causes b. Moist Heat Sterilization listeriosis (food poisoning). - ex. boiling or in b. Freezing autoclaves; effective at a - kills most bacteria, but lower temperature than survivors can remain alive for dry heat & it penetrates long periods in the frozen state; more quickly; bacteria cultures can be disadvantages of boiling - preserved by rapid freezing, does not kill thermophiles, sometimes with the addition of a endospores; autoclave is compound called DMSO, milk, or more effective than glycerol to protect proteins. boiling- it uses pressure to raise the temperature C. Radiation above that of boiling 1. Electromagnetic Spectrum - (121oC, 15psi, for 20 min.); Radiation used to sterilize liquids - classified by wavelength with ionizing and material easily and UV light radiation at the charred; used in food short-wavelength end, visible light in canning & the lab to the middle, & radio waves at the sterilize glassware & long-wavelength end. The shorter the media. wavelength, the greater its energy, & the more lethal it is. c. Pasteurization -Mode of Action: denatures DNA. - limits growth, but does not sterilize; used to slow Two types of radiation that kill bacteria spoilage of milk & dairy directly are UV (ultraviolet) Light & Ionizing products, wine, beer; Radiation. The effect of both is sterilization. advantage: causes a. UV Light minimal damage to the - bacteria actually have special product; developed by enzymes that can correct some Louis Pasteur; standard damage done by UV light!; in the  lab mercury vapor lamps - effect - stops microbial growth by (germicidal lamps) are used; stopping most chemical reactions (just disadvantages: kills only on like regular freezing) frequently used in surfaces & these wavelengths the microbiology lab to preserve can also be harmful to humans. perishable materials such as proteins, b. Ionizing Radiation blood products, & reference cultures of - 2 forms; both cause a chain of microbes ionizations by stripping electrons - used in the food industry to make from atoms, resulting in cell instant coffee, etc. death; disadvantages: - disadvantage - expensive. technically complex; is being used to sterilize some produce, F. Osmotic Strength much to the public's dismay. 1. Method - high concentrations of 1.) X rays salt or sugar. 2.) Gamma rays 2. Mode of action - microbes cannot grow if they are deprived D. Membrane Filtration of water; also, crenation or 1. Effect - physically removes shrinkage can occur (you're cellular organisms (not viruses - placing the microbes in a they are too small). hypertonic environment). 2. Uses - in lab, used with media, 3. Disadvantage - once added, antibiotics, & other heat solutes (such as salt or sugar) sensitive materials; filtration is cannot be easily removed; not replacing pasteurization in some used in the lab. cases, because filtration causes even less damage; you may have CHEMICAL METHODS OF MICROBIAL heard of the new "cold filtered" GROWTH CONTROL beers. SURFACTANTS E. Drying a. Structure - compounds with hydrophilic 1. Defined - the removal of water. & hydrophobic parts. Two processes: b. Mode of action - Penetrate oily a. evaporation involving heat substances in water & break them effect apart into small droplets that become - kills many microbes; rarely used in coated with surfactant molecules. The the lab, because the high heat causes hydrophobic end of the surfactant stick chemical changes (denaturation) into the droplets & the hydrophilic end -is used in the food industry. is attracted to the water. The result is b. Lyophilization [freeze drying] an emulsion, a fine suspension of oily - removes water directly by converting droplets in water, which can now be water from a solid state (ice) to a rinsed away. gaseous state; materials are frozen & c. Effect of soaps & detergents - wash placed in a chamber to which a partial away microbes, but do not kill them. vacuum is applied d. Wetting agents are surfactants that - avoids the chemical changes caused are often used with other chemical by heat drying agents to help the agent penetrate  fatty substances. Surfactants are not b. Mode of Action – when mixed with germicidal. water disrupt lipids in cell membranes e. Quaternary ammonium salts – four & denature proteins. organic groups attached to a nitrogen c. Ethanol & Isopropanol atom. - widely used as skin antiseptics; a 50 to 70% solution in water is the most Effect: kill all classes of cellular microbes & effective concentration (one of the few enveloped viruses by disrupting membranes. exceptions to the rule: increase effectiveness by increasing Uses: nontoxic & widely used in the home, concentration) industry, labs, & hospitals. Their - does not sterilize skin because it effectiveness is decreased in the presence of evaporates quickly and does not soap. Actually support Pseudomonas penetrate deeply enough into skin growth. Now being mixed with other agents pores. to overcome some of these problems. d. Main disadvantage - do not kill endospores. PHENOL & PHENOLICS a. Structure - compounds with hydroxyl HALOGENS groups (-0H) attached to a benzene a. Mode of Action - inactivates enzymes ring. by oxidation. b. Mode of Action - denature cell proteins, b. Examples disrupt cell membranes. 1.) Iodine – antiseptic c. Effect - kill most organisms; action is a) Tincture – iodine in a dilute not impaired by organic materials alcohol solution; one of first skin (remain active even in the presence of antiseptics. blood, feces, etc.) b) Iodophor – mixture of iodine and surfactants; ex. Betadine and Examples: Iodine (used for surgical scrubs 1.) Lysol and to prepare skin for surgery) 2.) Cresol – found in creosote; plant 2.) Chlorine – disinfectant; ingredient in derivative used to prevent the rotting household bleach; added to drinking of wooden posts, fences, railroad ties. water and swimming pools; inactivated 3.) Hexachlorophene - chlorinated by the presence of organic materials. phenolic; effective as an antiseptic; once widely used as an ingredient in HYDROGEN PEROXIDE soaps & lotions; in 1970's was found to a. Mode of Action – oxidizing agent increase risk of brain damage in (denatures proteins) babies; has now been replaced with b. Uses of H2O2: antiseptic for cleaning chlorhexidine in hospitals – good agent wounds, disinfect medical instruments for surgical scrubs. & soft contact lenses. When H2O2 comes into contact with tissue, it ALCOHOLS bubbles producing oxygen gas. This is a. Structure - compounds with a hydroxyl because all aerobes (incl. eukaryotes) group (-OH). produce the enzymes catalase & peroxidase which decompose H2O2  into oxygen & H2O. H2O2 generally b. Formalin - 37% solution. of kills microbes before it is destroyed by formaldehyde used to preserve tissues catalase or peroxidase. & to embalm; kills all microbes, including spores; lower concentrations You can differentiate between are used to inactivate microbes for Staphylococcus & Streptococcus using killed vaccines. H2O2; Staph is relatively resistant to H2O2 because of the large amounts of catalase & DYES peroxidase it produces.) May be used to - Ex. Crystal violet blocks cell wall clean deep puncture wounds, because the synthesis. oxygen produced kills obligate anaerobes - It effectively inhibits growth of G(+) present in the wound (ex. Clostridium). bacteria in cultures and in skin infections. HEAVY METALS - It can be used to treat yeast infections a. Mode of Action - heavy metals (mercury, copper, silver) react with the LESSON 4.2: ANTIMICROBIAL sulfhydryl groups of proteins CHEMOTHERAPY (denaturation) – term coined by Paul Ehrlich (father of b. Effect - kills many microbes. chemotherapy) c. Examples: - He discovered a drug treatment for syphilis; 1.) Mercuric chloride - once widely used as he also developed the guiding principle of an antiseptic; highly toxic; now chemotherapy, which is selective toxicity merthiolate & mercurochrome are used (the drug should be toxic to the infecting (less toxic); merthiolate is prepared as microbe, but relatively harmless to the host’s a tincture; use - basic first aid kit cells). Now the term chemotherapeutic agent supplies for disinfecting skin & mucous describes any chemical substance used in membranes. medical practice. 2.) Silver Nitrate - once applied to eyes of newborns to prevent gonorrhea; the ANTIMICROBIAL AGENT trend for a while was toward using – drug used to treat disease caused by antibiotics instead, but the microbes. development of antibiotic-resistant strains has necessitated the use of ANTIBIOTIC silver nitrate again. – a chemical substance produced by a 3.) Selenium sulfide – kills fungi, including microorganism that has the capacity to spores; commonly used to treat fungal inhibit the growth of bacteria and even skin infections; included in dandruff destroy them. shampoos (dandruff is often caused by - One of the first antibiotics was penicillin, a fungus). discovered by Alexander Fleming while he was carrying out experiments on ALKYLATING AGENTS Staphylococcus (1929). Some of his plates a. Mode of action - they alkylate (attach became contaminated with mold spores. As short chains of carbon atoms) to he examined them, Fleming noticed that the proteins and nucleic acids. Must not be Staphylococcus colonies were dissolving as used where they may affect human they neared the area where the mold was cells (these agents are carcinogenic). growing. -He reasoned that the mold was secreting - Penicillins – Bactericidal. Natural penicillins something that killed the bacteria. The mold are extracted from cultures of the mold was found to be a member of the genus Penicillium notatum. Penicillium, so he named the bacteria - Structure: contain beta lactam rings. The destroying substance “penicillin.” discovery of resistant bacterial strains led to - Ten years later Florey and Chain had the development of semisynthetic penicillins purified enough penicillin to begin (resistant bacteria have beta lactamase experiments involving the treatment of enzymes that can break down beta lactam humans. It was enormously useful in the rings). latter part of World War II. 2. Disruption of Cell Membrane Function GENERAL PROPERTIES OF - Antibiotics such as polymyxins act as ANTIBACTERIAL AGENTS detergents and distort cell membranes. A. Selective Toxicity - Polymyxins are obtained from Bacillus – drugs harm the microbe without causing polymyxa and are especially effective significant damage to the host. When against G(-) bacteria such as Pseudomonas searching for ways to treat disease, scientists that have phospholipids in their outer look for differences between the human (or membrane (along with the animal) host and the pathogen. Ex. Penicillin lipopolysaccharides). interferes with cell wall synthesis. Animal cells have no cell walls, so penicillin is not 3. Inhibition of Protein Synthesis toxic to animals. - Protein synthesis requires the DNA code, RNA (mRNA, tRNA, and rRNA). The B. Spectrum of Activity difference between bacterial and animal – the range of different microbes against ribosomes allows antimicrobial agents to which an antimicrobial agent acts. attack bacterial cells without damaging animal cells. Example: Broad spectrum: G(+) and G(-) - Ex. streptomycin, erythromycin, bacteria vs. Narrow spectrum: G(-) only. chloramphenicol 4. Inhibition of Nucleic Acid Synthesis - Target enzymes involved in nucleic acid synthesis (ex. DNA replication, transcription). 5. Interference of Metabolism Antimicrobial compounds can function in 2 ways: 1.) by competitively inhibiting enzymes and MODES OF ACTION 2.) by being erroneously incorporated into 1. Inhibition of Cell Wall Synthesis important molecules such as nucleic acids. - attach to enzymes that cross-link peptidoglycans. The actions of these compounds are sometimes called molecular mimicry because they mimic the normal molecule, preventing a reaction from occurring or causing it to go The procedure involves preparing several awry. dilutions of a chemical agent, inoculating them with the bacteria Salmonella typhi (a a. Competitive Inhibition digestive tract pathogen) or Staphylococcus - an antimicrobial compound binds to an aureus (a wound pathogen), incubating the enzyme’s active site, so that the enzyme tubes, and then checking for cloudiness in cannot react with its “true” substrate. the tubes, indicating growth. - To bind to the active site, the antimicrobial compound must be similar in structure to the The ratio of the effective dilution of the true substrate. chemical agent to the dilution of phenol that has the same effect is the phenol coefficient. b. Nucleic Acid Incorporation – Antimicrobial compounds such as A disinfectant with a phenol coefficient of 1.0 vidarabine and idoxuridine are erroneously has the same effectiveness as phenol. Less incorporated into nucleic acids. These than 1.0 means it’s less effective. Greater molecules are very similar in structure to the than 1.0 means it’s more effective. nitrogenous bases. - When incorporated into a nucleic acid, they 2.. Paper disc method – paper discs are garble the information that it encodes saturated with the chemical agent and because they cannot form the correct base placed on the surface of an agar plate pairs during replication and transcription. inoculated with a test organism. Clear “zones - These compounds can harm the host cells of inhibition” appear around the discs if the as well as the microorganisms (animal cells chemical agent is effective. use the same nitrogenous bases to make nucleotides). 3. Use-dilution test – The test microbe is - These agents are most useful in treating added to dilutions of the chemical agent. viral infections, because viruses incorporate The highest dilution that remains clear after these “fakes” more rapidly than cells and are incubation indicates a germicide more severely damaged. effectiveness. SIDE EFFECTS: ANTIBIOTIC SENSITIVITY TESTING A. Toxicity Antibiotic sensitivity testing plays a pivotal B. Allergy role in clinical pathology laboratories, aiding C. Disruption of Normal Microflora in the assessment of clinical isolates’ susceptibility to antibiotics. LESSON 4.3: TESTING This test can be conducted by placing small GERMICIDES paper disks soaked in antibiotics on agar 3 WAYS plates, with the resulting zone of bacterial 1. Phenol coefficients: Germicides can be growth inhibition serving as a measure of the tested by comparing their effectiveness to antibiotic effectiveness against the specific phenol, a traditional germicide. organism. It was phenol that Lister first used - he called Two primary method are used for conducting it carbolic acid. sensitivity tests: DISC DIFFUSION METHOD 4. On the basis of mode of action This routine method entails placing paper 5. On the basis of effects of their activity disks soaked in a specified quantity of 6. On the basis of route of administration antibiotic on agar plates containing pure cultures of the target organisms. The Chemical Structure antibiotics diffuse into the surrounding 1. Aminoglycosides: examples; medium, leading to the formation of visible Streptomycin clear zones, which are then measured and 2. Aromatic antibiotics (Nitrobenzene): compared to control results. eg. Chloramphenicol 3. Peptide antibiotics: eg. Polymyxin, PREPARATION Bacitracin, Gramicidin Filter paper disks are impregnated with a precise amount of antibiotic. Origin Inoculation: These prepared disks are then Microbial/Fungal/Actinomycetes carefully positioned onto agar plates that 1. Bacillus polymyxa: Polymyxin have been inoculated with a pure culture of 2. Penicillium notatum: Penicillin the microorganisms under investigation. 3. S. venezuelae: Chloramphenicol Antibiotic Diffusion: Over time, the antibiotics begin to diffuse from the disks into the Semi-synthetic antibiotics: surrounding agar medium. As a result, the Examples: Amoxicillin, Ampicillin, concentration of the antibiotic in the medium Doxycycline, Tigecycline, Sulfonamide etc increases near the disk. Synthetic antibiotics: Examples: Chloramphenicol (* it was extracted from Streptomyces venezuelae but now produced synthetically), 4-quinolones, Sulfonamide MODE OF ACTION Inhibitor of cell wall synthesis/ Peptidoglycan Inhibitors: Beta-lactam; Penicillin Bacitracin AGAR OR BROTH DILUTION METHOD Inhibitor of protein synthesis This more specialized method exposes Tetracycline organisms to varying concentrations of Mupirocin antibiotics in agar or broth. It provides more Inhibitor of Nucleic acid synthesis detailed information, particularly regarding Ciprofloxacin the minimum inhibitory concentration (MIC) Nalidixic acid of an antibiotic. Inhibitor of cytoplasmic membrane: Polymyxin; Colistin CLASSIFICATION OF ANTIBIOTICS Inhibitor of folic acid synthesis (Folate 1. On the basis of chemical structure antagonistic) 2. On the basis of origin Sulfonamide 3. On the basis of range of activity ( spectrum of activity) EFFECTS OF THEIR ACTIVITY Bactericidal: Kills bacteria Examples: Aminoglycosides, Penicillin, Cephalosporin Bacteriostatic: Inhibits the growth of bacteria Examples: Sulfonamide, tetracycline, chloramphenicol, trimethoprim, macrolides, Lincosamide ROUTE OF ADMINISTRATION Oral antibiotics: Acid stable antibiotics, Examples; Penicillin V Parenteral route: Intravenous administration Examples; Penicillin G GRAM’S IODINE CRYSTAL VIOLET STAINING REAGENT GRAGRAM