Microbiology: Controlling Microbial Growth In Vitro PDF

4) Gaseous atmosphere: à Gases mostly linked to microbial growth are: Classified according to presence of protective enzymes...

4) Gaseous atmosphere: à Gases mostly linked to microbial growth are: Classified according to presence of protective enzymes 1) Obligate aerobes: Require O2 in atmospheric concen (21 %). Microbiology Controlling Microbial Growth In Vitro 2) Microaerophiles: Require O2 in subatmospheric concen (5 %). 3) Facultative Grow both in presence or absence of O2 but anaerobes: growth is better in its presence: à Factors That Affect Microbial Growth o O2à Aerobic respirationà better. 1) Availability of nutrients: o No O2à fermentation à low ATP but § Nutrients serve as sources of energy as well as chemical elements. enough. § Amount of elements needed differs according to the type of element: o Micronutrients: Mg , Ca, Na , Fe, K, Cl, & I O2 4) Aerotolerant Grow in the presence of O2, but no better o Macronutrients: Major elements : C,H,O,N,P,S anaerobes: than in its absence: o Some trace elements o Doesn’t generate extra ATP from O2. o Protect itself from ROS by protective enzymes. 2) Potential of Hydrogen (pH): à Classified according to their preferable pH of growth into: à Two possibilities: § Alkaliphiles: prefer a pH more than 8.5 (Vibrio cholerae). 1) Very goodà Growth in presence= absence § Most microbes prefer a neutral or slightly alkaline medium: 2) Goodà Growth in absence is better o pH between 7 and 7.4 5) Obligate Only grow in the absence of O2. o Human PH : Most microbes prefers Human PH: a lot of IF anaerobes: § Acidophiles: prefer a pH between 2 and 5 (H. pylori & fungi). CO2 Capnophiles Require CO2 in high concen (5-10 %). 3) Moisture: à Thioglycolate broth § Water is essential for life as cells are 70 to 95% water. § Supports the growth of all bacteria § Life forms are able to survive even through desiccation § Used to increases number of MOs. (complete dryness): § Obligate aerobesàobligate anaerobe. o Bacterial spores. § Grows in part matches its requirment. o Protozoan cysts. 5) Atmospheric pressure: § Piezophiles : able to grow under high atmospheric pressures. 6) Osmotic pressure and salinity: Bacterial Culture Media (Artificial media) § Osmotic pressure: exerted on the cell membrane by the solutions. à Classification according to physical nature: § Osmosis: movement of the solvent through a semipermeable 1) Broths media (liquid or tube media) membrane from a hypotonic solution to a hypertonic solution. o If sample has more than one bacteria à will mix with each other § Halophilic MOs: prefer salty environments for growth o No sperationà cannot isolate MOs. § Haloduric MOs: don’t prefer salty environments, but are able to grow § Plasmolysis: shrinkage of cytoplasm & detachment plasma 2) Agar (Solid media) membrane from the cell wall due to water loss o Complex polysaccharide obtained from a red marine alga § Plasmoptysis : bursting or rupture of cell when excessive water o Used as a solidifying agent (like gelatin) o Sample has more than one bacteria à not mix with each other. Solution Type Cells with CW Like bacteria Cells without CW o Sperationà can isolate MOs. Like RBCs Hypertonic Plasmolysis Crenation (shrink) à Classification according to culture contents: Isotonic No effect No effect Hypotonic Plasmoptysis Hemolysis 1) Chemically defined medium o All ingredients are known. o Medium was prepared in the laboratory. 2) Complex medium o One in which the exact contents are not known. o Contain ground-up or digested extracts from animal organs o Know approximate composition but not the exact composition. o Provide the necessary nutrients, vitamins, and minerals. 7) Temperature: à Enriched ,selective & Differential medium: § Thermophiles: prefer high temperatures. 1) Enriched medium: § Hyperthermophiles (Extreme thermophiles): prefer temp above 100°C § Containing a rich supply of special nutrients § Mesophiles: prefer moderate temperatures. § Promotes the growth of fastidious organisms. § Psychrophiles: prefer cold temperatures. § Fastidious organism: need special mixture(complex) of AA & § Psychrotrophs: Prefer temperature around 4°C for growth vitamins (different FMOs need à different Enriched Medium) (refrigerators ’’food spoilage’). § Prepared by adding extra nutrients to a medium called nutrient agar. § Psychroduric: tolerate cold temperatures, but prefer warmer temp. § Examples of solid enriched media: Category Minimum GT Optimum GT Maximum GT Blood agar Chocolate agar (°C) (°C) (°C) § Bright red. § Brown : heating blood agar. Thermophiles 25 50–60 113 § Nutrient agar + 5% sheep RBCs § Hemoglobin is more readily Mesophiles 10 20–40 45 accessible. Psychrophiles –5 10–20 30 § More enriched than blood agar  2) Selective medium: MEDIUM PICTURE § Added inhibitors that discourage the growth of certain organisms without inhibiting the growth of the organism being sought. § Allow microbe A to grow but inhibt microbe B (by antibiotics) § Example: Medium Selective For Inhibits MacConkey Agar Gram - bacteria Gram + bacteria Phenylethyl Alcohol (PEA) Gram + bacteria Gram - bacteria Blood agar Chocolate agar Colistin-Nalidixic Acid Thayer-Martin Agar N.gonorrhoeae Other bacteria (using Martin-Lewis Agar antimicrobial agents) Salt-enriched Medium Salt-tolerant Non-salt-tolerant (haloduric) bacteria bacteria 3) Differential medium Mannitol Salt agar § Permits the differentiation of organisms that grow on the medium. § Gives each microbe specific characteristics § Allow Microbe (A+B) to grow but in different apperance. § Example: Agar Type Characteristics MacConkey § Differentiates B/W GNB : o Lactose-fermenting: pink. o Nonlactose-fermenting: colorless. MacConkey agar Blood agar Blood § Determine the type of hemolysis: o Non-hemolytic: Gamma hemolytic streptococci Summary o Partial hemolysis (green zone): Alpha hemolytic à Categories of media are not mutually exclusive: o Complete hemolysis (clear area): Beta hemolytic o Blood agar is enriched and differential. Mannitol § Used to screen for S. aureus: o MacConkey agar and MSA are selective and differential Salt o Fermenter: originally pink medium turns yellow o PEA and CNA are enriched and selective Incubators: Microbial population § Lab equibment provide approprite gas concen + apporpiate temp: 1) Ordinary incubators: Contain room air (21% O2 + 1% CO2 + 37 °C temp) 2) Anaerobic incubator: No O2 à anerobic bacteria 3) CO2 incubator: Co2 5-10%à capnophiles Bacterial growth § Growth refers to an increase in number of cells through binary fission. § Generation time: The time it takes for one cell to divide into two. § GT increase à Slow growers & viceversa: o Rapid growers: § Shows changes in concentration of viable microbesà Y-axis Staphylococcus spp & Streptococcus spp. (20 min) § Shows changes in character with timeà X-axis. Pseudomonas spp. & Clostridium spp. (10 min) à It consists of the Four phases: o Slow growers: Mycobacterium spp 1) Lag(delay) phase: o Bacteria get prepared for cell division without increasing in à Colony: number. 2) Log (exponential) phase: o Macroscopic accumulation of same MOs o Colony forming units (CFU) : o Bacteria divide rapidly and their number increases exponentially. § Original single bacteria cells that lead to form colony. o Growth rate is the greatest during the log phase o Million of MOs in agarà no movment à seen by naked eye 3) Stationary (constant)phase: o By appereance of colony can defferantiate B/W: o Number of viable bacteria becomes constant. § Pure culture: contain similar colonies (only one species) o Number of bacteria forming equals number of bacteria dying. § Mixed culture: more than one species ( different colonies à o Greatest population density in this phase. different appereance) 4) Death (Decline) phase: o Number of viable bacteria declines rapidly as they die in large à Sterile techniques: numbers. o Overcrowding occurs& nutrient supply decreases. o Used to prevent other environmental microbe to grow in culture with o Concentration of toxic waste products continues to increase MOs in the sample o This stage also witnesses morphological changes such as: o Only sample MOs in culture. § Formation of L-forms (pleomorphism : deffictive CW) § Formation of spores. Chemostat 3) Protozoa: § Used many industrial and research procedures § Maintain of continious supply of certain MOs § Protozoal cultures are usually reserved for research & reference labs. § Keep MOs in log phase. § CML attempt to culture amebas & ameba-like organisms: § Maintenance of an essential species of microorganism. o Acanthamoeba spp. § Chemostat regulates: o Naegleria spp. 1. Protozoa (tiny organisms) are usually grown in special research labs, not in regular diagnostic labs. 2. 3. CML (Clinical Microbiology Laboratory) may try to grow certain types of protozoa, like Acanthamoeba and Naegleria, which can cause serious brain infections. Growing these protozoa is expensive and difficult. o Causes fatal CNS infections. 4. There are two ways to grow these protozoa in the lab: Axenic culture: This means growing the protozoa without any other cells or microorganisms. o Supply of nutrients and sterile air. Xenic culture: This means growing the protozoa with other cells, like bacteria, to help feed them. 5. Sometimes, cell cultures or bacteria are used as food for the protozoa when growing them in the lab. o Removal of waste products and excess microorganisms. § This may be done using: o Axenic: doesn’t contain foreign cell o Xenic: § Contain foreign cell. § Using cell cultures or bacteria as a food source for protozoa Inhibiting the Growth of Microbes In Vitro § Sterilization: complete eradication of all living organisms (include all Microbes that need specific requirements for lab growth, such as: life forms). 1) Obligate intracellular pathogens: § Disinfection: elimination of pathogens on nonliving objects. § Viruses, Chlamydia spp, Rickettsia spp. § Disinfectant: chemical substance used to eliminate pathogens on § Cultured in cell cultures , embryonated chicken eggs or lab animals. nonliving objects. § Cell cultures specific lines :monkey kidney cells, human lung cells, § Antiseptic: chemical substance used to eliminate pathogens on living cancer cells surfaces: § Positive growth is indicated by specific morphological microscopic changes (cytopathic effects). o Skin § Used in CMLàtime consuming àPCR & serology test is alternative o Mucous membranes. § Antiseptic technique: is the use of antiseptics to fight pathogens on 2) Fungi: living surfaces. § Cultured on such media as: § Aseptic technique: is the use of a method (physical or chemical) to o Brain-heart infusion (BHI) agar. fight microbes while carrying out a procedure in the healthcare setting. o Sabouraud dextrose agar (SDA). § Pasteurization: eliminate pathogen in milk (liquid) § Selection for fungal growth against bacteria is provided by o Not a sterilization but disinfectant procedure o Adding antibacterial agents. o Reducing the pH § Sanitaization: § Care must be taken in the lab against the spread of potentially o Reduction of microbial populations in public health setting infective spores. (in community) Degree of Resistance of Microbes to Disinfection and Sterilization Physical Methods to Inhibit Microbial Growth Level of Resistance Microbes 1) Heat: High § Prions. § The combined importance of temperature and time § Bacterial spores. § Assessed by: § Coccidia (forms of protozoa). o Thermal Death Point (TDP): Lowest temp that kills all MOs in § Mycobacteria. a standardized pure culture within a specified time. Intermediate § Nonlipid (naked viruses). o Thermal Death Time (TDT): time required to sterilize a pure § Extremely small viruses culture at a specified temperature. § Fungi § MOs that has high TDP & TDT are more resistant. Low § Vegetative bacteria § Lipid or medium-sized viruses. à Divides according to concomitant absence or moisture use into: A) Dry heat: o Mainly used for objects which can withstand high temperatures. Cidal & static o It is usually performed at: § 160-165°C for 2 hours. § Static : inhibit microbial growth without killing it. § 170-180°C for 1 hour. § Cidal : microbes killing: o Incineration (‫) اﺗﻼف ﺑﺎﻟﺤﺮق‬: used to burn & completely destroy Agent Type Definition contaminated, disposable objects. Germicidal, General terms meaning killing o Flaming is another dry heat method used to achieve sterility. Biocidal or microbes microbicidal agents Bactericidal Kills bacteria, but not necessarily B) Moist heat: bacterial endospores o Achieves results at lower temperatures than dry heat. Viricidal Kills viruses o Application of moist heat may take the form of: Fungicidal Kills fungi, including fungal spores 1) Boiling: which kills most vegetative microbes after 30 minutes. Algicidal Kills algae 2) Autoclaving: Sporicidal Kills bacterial endospores (Sterilization) § Practical method for achieving sterility in healthcare settings. § Used: Pressure + heat + moisture to enhance killing effect. Pseudomonicidal Kills Pseudomonas bacteria § Performed at 121.5°C and 15 psi for 20 minutes, Tuberculocidal Kills Mycobacterium tuberculosis § Care must be taken to allow steam to adequately penetrate the material in question. § Indicators : to ensure the necessary conditions have been met. 2) Cold: Properties of the ideal disinfectant: § Refrigeration slows down bacterial growth. 1) Easy to prepare and apply. § Slow freezing kills bacteria (by ice crystal). 2) Soluble in water & broad antimicrobial spectrum. § Rapid freezing keeps bacteria in suspended animation, making it 3) Fast-acting. useful in preserving bacterial cultures. 4) Not affected by organic matter. 3) Desiccation: 5) Nontoxic to human tissue. § Removes the moisture necessary for bacterial growth. 6) Nondestructive to inanimate objects. § Lyophilization : process combining the effects of cold and drying to 7) Leave a residual antimicrobial film. preserve a wide variety of objects. 8) Stable, Odorless & Inexpensive. 4) Radiation: à As there is no ideal disinfectant: § Non ionizing radiation: o Power of a certain disinfectant is assessed by comparing it to that o Use of UV light damages microbial DNA, leading to death. of phenol using the phenol coefficient test o UV lamps may be used in a wide variety of settings o One must take care as they may cause skin and eye damage. § Ionizing radiation; Mechanisms of action of disinfectants: o Use of radiations with shorter wavelengths (high energy), 1) Destruction of cell membranes: o Such as X-rays and gamma rays (made with more caution) o Surface-active soaps and detergents 5) Ultrasonic waves: Transmit energy to substances stuck on object o Alcohols. surfaces, thus facilitating their removal. o Phenols. 6) Filtration: Used in settings where the use of heat is inappropriate like 2) Destruction of structural proteins and enzymes: protiens. o Hydrogen peroxide. 7) Gaseous atmosphere: o Alcohols, phenols. o Formaldehyde, halogens (hypoclorides, iodines & florides) § Modifying gas concen in environment may make it less appropriate o Heavy metal salts (silver, nickel , & mercury). for the growth of certain microbes, § Depending on their specific O2 and CO2 requirements. 3) Attacking nucleic acids :ethylene oxide: § Hyperbaric oxygen theraby: used to kill obligate anaerobes. Chemical Methods for Inhibiting Microbial Growth à Factors considered when evaluating a disinfectant: 1) Concentration of the disinfectant (efficieny increases with concen). 2) Physical nature of the object to be disinfected. 3) Prior cleaning of the object to be disinfected from organic matter. 4) Bioburden: is the type and quantity of the microbe to be eradicated. 5) Contact time , temperature & pH.