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Bacterial Cultivation (Culturing) Function of Clinical Microbiology Lab • Participation in management of patients with infectious diseases by processing of clinical specimens: 1- Microscopy. 2- Cultivation & isolation of pathogens. 3- Identification of isolates: macroscopy, microscopy, biochemical testing & serology. 4- Antimicrobial susceptibility testing (AST) of isolates. Bacterial Cultivation Definition & Purposes • Collection of bacteria-containing clinical specimens from patient’s infection site (i.e. in vivo) & growing these bacteria in artificial Lab bacteriological culture media (i.e. in vitro) → isolation of dispersed (individual), significant (↑ number), pure bacterial colonies → macroscopic examination of colony characteristics on primary culture media → early preliminary information regarding bacteria isolated → subsequent laboratory confirmatory procedures → definitive characterization & identification and Antimicrobial Susceptibility Testing (AST) → interim & final reporting to the clinician. Bacterial Cultivation Growth Requirements • The appropriate growth requirements for in vitro bacterial cultivation include: 1- Nutritional needs in culture media. 2- Environmental/ incubation conditions. Growth Requirements 1- Nutritional needs • Water: used to dissolve materials to be transported across the cytoplasmic membrane. • Source of carbon (glucose): required for the construction of all organic molecules. • Source of nitrogen: obtained either inorganic or organic source. • Source of minerals. • Buffer System. Growth Requirements Nutritional needs • Nutrients as blood, milk, yeast extract & beef extract are incorporated into culture media. • Pathogenic bacteria are classified into: Fastidious (needs are relatively complex, with exceptional media components) vs Nonfastidious (needs are relatively basic & straightforward). • various types of culture media are used in Microbiology lab according to different needs of pathogenic bacteria. Growth Requirements 2- Environmental/ Incubation conditions • Four environmental (incubation) conditions to support the growth of clinically relevant bacteria include: Oxygen & carbon dioxide (CO2) availability. Temperature. pH. Moisture content of medium & atmosphere. Oxygen & Carbon dioxide (CO2) availability Oxygen (O2) • Most clinically relevant bacteria are either: • Aerobic: use oxygen as a terminal electron acceptor & grow well in room air. • Facultatively anaerobic: being able to grow in presence (i.e. aerobically) or absence (i.e. anaerobically) of oxygen. • Strictly anaerobic: are unable to use oxygen as an electron acceptor. Oxygen (O2) • some bacteria, as Pseudomonas spp., members of Neisseriaceae family, Brucella spp., Bordetella spp., & Francisella spp., are strictly aerobic & cannot grow in absence of oxygen. • Other aerobic bacteria require only low levels of oxygen & are referred to as being microaerophilic, or microaerobic. Carbon dioxide (CO2) • Capnophilic organisms grow best with higher CO2 concentrations (i.e. 5-10% CO2) than is provided in room air. • For some bacteria, 5-10% CO2 concentration is essential for successful initial cultivation from clinical specimens. Temperature • most medically important bacterial pathogens generally multiply best at temperatures similar to those of internal human host tissues & organs (i.e. 37°C) → incubators with temperatures 35°-37°C are used for cultivation. • Incubation at certain temperatures can be used as an enrichment procedure for enhanced recovery of certain organisms: Campylobacter jejuni grows at 42°C, whereas many other pathogens & non-pathogens cannot. Cold enrichment for Listeria monocytogenes & Yersinia enterocolitica at 0°C, however they grow best at temperatures between 20° & 40° C. pH • Most clinically relevant bacteria prefer a near neutral pH range (6.5-7.5). Moisture • Loss of water from media can be deleterious to bacterial growth in two ways: less water is available for essential bacterial metabolic pathways . relative increase in solute concentration of the media. • Measures to ensure that appropriate moisture levels are maintained throughout the incubation period include: sealing agar plates to trap moisture. using humidified incubators. Culture media • Culture medium or growth medium is a liquid or gel designed to support the growth of bacteria. Culture Media Physical Status 1- Liquid (Broth): contains NO agar. 2- Solid (Agar): contains 1.5% agar. (N.B. Biphasic medium contains liquid & solid parts e.g. Brucella Castaneda medium). 3- Semisolid: contains 0.5% agar e.g. Motility medium. Culture Media Broth media • nutrients are dissolved in water. • Used 1- for growth of pure batch cultures e.g. Nutrient broth & 2- as Enrichment broth e.g. alkaline peptone water for Vibrio cholera. • Growth is indicated by change in appearance from clear to turbid (i.e. cloudy). • The more bacterial growth, the greater the broth’s turbidity. • At least 106 bacteria/mL of broth are needed for turbidity to be detected with unaided human eye. Culture Media Solid media • made by adding solidifying agent to nutrients & water. • Agarose: polysaccharide extracted from seaweeds, most commonly used solidifying agent, No nutritive value, NOT affected by growth of bacteria, & melts at ≥95°C but resolidifies at <50°C → allows for heating to extremely high temperature required for sterilization, cooling to 55-60°C for distribution into petri dishes and tubes, & on further cooling, forming stable solid gel (Agar). Culture Media Solid media • Three forms: 1- Agar plate (petri dishes): provide large surface area for culture. Incubated in inverted position (agar-compartment upwards & dish-cover downwards). 2- Agar Slant (+ butt): e.g. Triple sugar iron. 3- Agar Deep: used for storage & study of gaseous requirements of bacteria. Culture Media Agar plates Nutrient agar Blood agar Preparation of Culture Media General • Broth & agar media are prepared by reconstituting (dissolving) specified amount of powder in water (distilled or deionized). • Boiling is often required to dissolve the powder. • Most media require sterilization to allow only growth of bacteria from patient specimens & prevent growth of contaminants from water or powdered media. Preparation of Culture Media Additives • Other sterile supplements as sheep blood or specific vitamins, nutrients, or antibiotics, should be added when molten agar has cooled, just before distribution to plates. • Delicate media components that cannot withstand steam sterilization by autoclaving (e.g. serum, certain carbohydrate solutions, certain antibiotics, & other heat-labile substances) can be sterilized by membrane filtration: Passage of solutions through membrane filters with pores 0.2-0.45μm in diameter. This will effectively remove most bacterial & fungal contaminants but NOT viruses. Preparation of Culture Media Agar Plate Medium • • • • Weight dehydrated media. Dissolve in distilled H2O to 1L volume. Adjust pH to 7.0 Boil media then distribute in bottles or large flasks covered with plastic screw caps or aluminum foil respectively. • Sterilize for 15 minutes using autoclave or pressure cooker at 121°C under 15 Ibs. • Cool to 50°C, & pour molten agar into individual petri plates. Preparation of Culture Media Tube Medium • • • • • Weight dehydrated media Dissolve in distilled H2O to 1L volume. Adjust pH to 7.0 Boil media then distribute in individual test tubes. Sterilize for 15 minutes using autoclave or pressure cooker at 121°C under 15 Ibs. • Cool to 50°C, & pour into individual tubes: For slant agar, place tubes in slant position until cool. For broth media or deep agar, place tubes in upright position until cool. 1- Assemble all chemicals in work area before you begin 2- Accurately weigh each of the dry ingredients in your culture media 3- Add each dry culture medium ingredient to the culture flask 4- Add distilled (or deionized) water to make the correct volume. Heat & stir (agar will burn if it is NOT stirred) until all of the ingredients go into solution. When the media boils, it is ready for sterilization. 5- Media sterilization 6- Sterilize for 15 minutes using the wet cycle (autoclave) or at maximum pressure in a pressure cooker. Remember to cover the top of the flask or jar with aluminum foil to prevent contamination when the media cools. N.B. When using a pressure cooker, don’t over fill the cooker! 7- Line your sterile petri plates along the edge of the table. Transfer hot media to small sterile container & pour 15-20 ml of culture media into each petri plate. The petri plate lid should be open slightly, but NOT completely open as this increases contamination. The Petri Porter 11-May-17 NM Kaplan 35 Culture Media Types • Four general categories of according to their function & use: Supportive. Enrichment. Selective. Differential. media Culture Media Supportive • contain nutrients that • Nutrient agar plate support growth of most non-fastidious organisms without giving any particular organism a growth advantage. • Examples: Nutrient agar & Nutrient broth. Culture Media Enrichment • Contain nutritive substances like blood, serum, or egg → grow most bacteria e.g. Blood agar & Chocolate agar. • Some contain specific nutrients → grow particular bacterial pathogens that may be present alone or with other bacterial species in patient specimen e.g. Buffered charcoal-yeast extract (BCYE) agar that provides Lcysteine & other nutrients required for growth of Legionella pneumophila (causative agent of legionnaires’ disease). • Enrichment broth: used to enhance the growth of particular bacterial pathogen from mixture of organisms e.g. selenite F broth (Salmonella species) & alkaline peptone water (Vibrio cholera). Blood agar Chocolate agar Growth of Legionella pneumophila on BCYE enrichment agar Culture Media Selective • contain one or more agents that inhibit the growth of all unwanted organisms except those being sought. • Inhibitory agents used include dyes, bile salts, alcohols, acids, & antibiotics. • Example: MacConkey agar & SalmonellaShigella agar. Culture Media Differential • contain an indicator as pH indicator that allows colonies of one bacterial species to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria growing on the same agar plate. • Examples: MacConkey agar that differentiates between lactose-fermenter (LF) & non lactose-fermenter (non LF) Gram-negative bacteria. Blood agar & different hemolytic patterns. Culture Media Many with more than one function • Selective & differential: MacConkey agar: Gram negative bacilli as LF vs. NLF. Mannitol Salt agar (MSA): Staphylococci. Hektoen enteric (HE) agar & Xylose-lysinedesoxycholate (XLD) agar. • Enrichment & differential as sheep blood agar which contains general nutrients & 5-10% sheep blood → different hemolytic patterns of Streptococcus species colonies. MacConkey agar • Selective for Gramnegative bacilli due to bile salts & crystal violet that prevent growth of most Gram-positive bacteria. • Differential due to lactose (sugar) & pH indicator neutral red: • Lactose fermenter; acidic pH; deep purple colonies. • Non-lactose fermenter; alkaline pH; colorless colonies. Mannitol Salt Agar • Selective for staphylococci due to high salt (NaCl) concentration (7.5%). • Differential due to mannitol (sugar) & pH indicator phenol red: • Mannitol fermenter; acidic pH; yellow colonies; S aureus. • Mannitol nonfermenter; red colonies; alkaline pH; S epidermidis & S saprophyticus. Different hemolytic patterns on Sheep Blood agar 1- Beta hemolysis: complete lysis of RBCs → colorless zone surrounding the colony. 2- Alpha hemolysis: zone of partial hemolysis surrounding the colony, often accompanied by greenish discoloration of agar. 3- Gamma reaction: NO hemolysis or discoloration of agar surrounding the colony. Bacterial Cultivation Tools • Cotton swabs. • Plastic pipettes. • Loops & wires/ needles: 1- Plastic. 2- Metallic: made of platinum or Ni-chrome; sterilized by Flaming = incineration of all life forms, which begins from handle towards the tip to prevent bacteria from forming aerosols (= water droplets of live bacteria sprayed into air). Bacterial Cultivation Tools Bacterial Cultivation Culturing Methods in Broth medium • by direct inoculation of clinical specimen using cotton swab or plastic pipette. on Agar plate 1- Streak-plate method: (commonest & routinelyused). 2- Pour-plate method. 3- Spread-plate method. (2 & 3 uncommonly used). 1- Streak-plate method 1- direct inoculation of one loop-full of the specimen onto small peripheral area of the surface of well dried agar plate. 2- Streaking in a standard pattern to distribute inoculum thinly in a series of parallel lines in different segments of the plate using loop (with flaming between different series) → even spreading over the entire surface. 3- Following incubation, separated colonies are obtained over the last series of streaks. Inoculation & Streaking with flaming Semi-quantitative culturing using calibrated loop streaking technique = inoculation of measured amount of liquid specimen as urine for enumeration of bacterial colonies (colony-forming units; CFUs). Streaking Pattern Well-isolated dispersed separated colonies Contamination 2- Pour-plate method 3- Spread-plate Method Bacterial Cultivation Subculturing • Transfer of microorganisms from one media to another. • Purposes: 1- Isolation of pure culture from mixed one. 2- Performance of microbiological test procedures. 3- Preparation & maintenance of stock culture. Isolation of pure culture from mixed one Picking Colonies General Aseptic Precautions 1. Sterilize the bench working area before & after use. 2. Sterilize metallic inoculation loop or needle before & after use. 3. Work quickly & never place the tube’s plug on bench. 4. Flame the lips of the tube before & after work. 5. Never leave the tube open any longer than the time needed to transfer the culture. 6. The container must be covered to prevent entrance of microorganisms.