Lecture 9: Culture Media & Methods PDF

LECTURE 9 Culture Media & Methods Ms. Peters  Culturing Microorganisms There are five basic techniques to manipulate, grow, examine and characterize microorganisms in the lab Inoculation Incubation Isolation Inspection Identification...

LECTURE 9 Culture Media & Methods Ms. Peters  Culturing Microorganisms There are five basic techniques to manipulate, grow, examine and characterize microorganisms in the lab Inoculation Incubation Isolation Inspection Identification (the five I’s) Aids in handling and maintaining of microorganisms as discrete entities  Culturing Microorganisms Inoculation: – The action of introducing the microorganism into fresh medium is called inoculation – A small amount microorganisms (an inoculum) is introduced into culture medium – The culture medium provides the appropriate environment that supports the growth and metabolism of the organism – Observable growth on the medium are referred to as colonies  Culturing Microorganisms Incubation: The process of providing the required growth conditions and allowing microorganisms to grow on media is known as incubation Atmospheric conditions can be aerobic, anaerobic or with increased CO2 Growth temperature: RT -30ºC, 35-37ºC, 42ºC Length of incubation: 18-24 hrs, 7-10 days, 4-8 weeks Inoculation is the introduction of microorganisms to media while incubation is allowing microorganisms to grow under supplied growth conditions.  Culturing Microorganisms Isolation: The primary method to separate different groups of microorganisms. It allows separation of different groups of bacteria based on the growth pattern Since it is formed from a single cell, the colonies formed are thought to be of one bacterial species Isolation methods include: – Streaking Method – Pouring Method – Spreading Method – Serial Dilution Method  Culturing Microorganisms Isolation Methods Streak Plate Method Pour Plate Method Spread Plate Method Dilution Method Mix Plate Pure Plate  Culturing Microorganisms Culture Media – Culture media are contained in tubes and petri dishes – They are inoculated with sterile loops, needles, pipettes and swabs – Media varies widely in nutrient content and consistency – Culture media can be classified on three primary levels: 1. Physical form 2. Chemical Composition 3. Function  Culturing Microorganisms Culture Media – Physical form: Liquid Water - based solutions that do not solidify at freezing temperatures or evaporates at high temperatures Growth occurs through out the media and can be identified as the medium becoming turbid or cloudy. E.g., Alkaline Peptone Water, Nutrient Broth, selenite F Broth  Culturing Microorganisms Culture Media – Physical form: Semi- solid Media with clot like consistency due to the presence of solidifying agent ( 0.3 – 0.5% agar) Commonly used to determine motility of organisms and the preservation of stock cultures E.g., Sulfide Indole Motility (SIM)  Culturing Microorganisms Culture Media – Physical form: Solid – Media that provides a firm surface for growth of cells due to the presence of agar (1 – 5%) – It is solid at room temperature but melts at 100⁰C – Resolidify when cooled to 42⁰C – E.g., Blood agar, chocolate agar, Nutrient agar Blood Agar Chocolate Agar Nutrient Agar  Culturing Microorganisms Culture Media – Chemical Composition: The composition of the media reflects the natural surroundings of the microorganism. It may depend on the function of the medium Based on chemical composition, media can be: – Defined (Synthetic) – all chemical components are known E.g., Basal Salts medium – Complex Media – media that has at least one chemical component is unknown. E.g., Nutrient Broth  Culturing Microorganisms Culture Media – Function: General Purpose Media – Designed to support the growth of a broad spectrum of microbes. – Contains a mixture of nutrients that can support both pathogenic and non - pathogenic microbes – E.g., Nutrient agar, Nutrient Broth Enriched Media – Supports the growth of fastidious organisms – It’s a basal growth medium with additive nutritive supplements such as blood, serum, specific vitamins etc. – E.g., Blood agar, Chocolate agar, Thayer-Martin medium  Culturing Microorganisms Enriched Media – Composition :- Nutrient agar + 5% Sheep blood – Uses :- Routine culture and hemolytic property of bacteria  Culturing Microorganisms Culture Media – Function: Enrichment Media – Usually, liquid media that suppresses the growth of commensals – Provides additional nutrients to allow certain microbes to flourish – E.g., Alkaline Peptone Water (APW), Selenite F Selective Media – Contains an agent that inhibits the growth of one type or group of microbes an allowing the other to flourish – Important in the primary isolation of a specific type of microorganism from samples containing several different species such as stool – E.g., Mannitol salt agar (MSA), Eosin Methylene Blue (EMB), – Thiosulfate citrate bile salt sucrose agar (TCBS ) – Deoxycholate citrate agar (DCA)  Culturing Microorganisms Enrichment Media Selenite- F broth Alkaline peptone water: Composition: Peptone water pH 8.6 Composition: Peptone water + sodium Uses: Enrichment medium for selenite Vibrio spp. Uses: Enrichment medium for salmonella  Culturing Microorganisms Selective Media Thiosulphate Citrate Bile salt sucrose agar (TCBS) Composition: Thiosulphate + Citrate + Bile salt + Sucrose + Bromothymol blue Uses: Culture of Vibrio cholera  Culturing Microorganisms Culture Media – Function: Differential Media – Supports the growth of several different type of bacteria – Designed to produce visible differences between the bacteria due to the presence of pH indicators – E.g., MacConkey Agar, Urease Media NOTE: Indicator Acid Alkaline Phenol red Yellow Pink Neutral red Pink/red Yellow Bromothymol Blue Yellow Blue Transport Media – Used to maintain and preserve microorganisms in transit until it is able to be cultured – It does not promote growth but rather maintains viability – E.g., Cary – Blair Media, Stuart’s and Amies Media  Culturing Microorganisms MacConkey (Differential media) Composition :- Peptone + Lactose+ Sodium taurocholate + Neutral red + Agar Uses :- Culture of Gram - negative bacteria (differentiate lactose fermenting from non lactose fermenting bacteria).  Culture methods – In order to grow bacteria on culture media, various techniques can be used – Such techniques include: » Streak Culture – For isolated colonies » Lawn Culture - Uniform- Drug Sensitivity » Stroke Culture - For Tubes for pure growth slide agglutination » Stab Culture - Stabbing medium in tubes- with long straight wire » Anaerobic Culture - Anaerobic jar :- McIntosh-Slides Gas pack system  Culture Methods – Streak culture Four quadrant technique used for the isolation of pure culture of the microorganisms from a mixed population. The inoculum is streaked in a way that bacterial cell density is diluted to produce isolated single colonies. Culture Methods Culture Methods  Culture Methods Stab Method: By puncturing a suitable culture media (nutrient agar) with a long straight charged wire. For gelatin liquefaction, stock cultures and motility.  Culture Methods Anaerobic Culture - McIntosh –Slides Anaerobic jar It is a stout glass or metal jar with lid which has an inlet for gas and outlet. The lid contains a catalyst for e.g., Alumina pellet coated with palladium. Inoculated plates are kept inside the jar Air is removed and lid is clamped tight  Culture Methods Antibiotic Susceptibility Testing Methods Used to determine a microbe’s vulnerability to antimicrobial drugs by exposing a standardized concentration of organism (0.5 McFarland) to specific concentrations of antimicrobial drugs. Testing can be done qualitatively, semi quantitatively, or using nucleic acid–based methods. Methods include: Disc diffusion Broth dilution (M.I.C.) E-test Nucleic acid based methods  Culture Methods Disk Diffusion (Kirby- Bauer) Results are usually reported as one of the following: – Susceptible (S) – Intermediate (I) – Resistant (R) Zones of inhibition are measured and compared to an standard (usually CLSI breakpoints)  Culture Methods E- Test Method » Combination of agar diffusion and MIC calibrations » Strips contain measured amount of antibiotic at end » Scale along length of strip » View growth inhibition » Read on scale to get MIC  Culture Methods Broth Dilution (MIC) » Oldest method of antimicrobial susceptibility testing method ( macrodilution) » A two-fold dilution of antibiotics (E.g., 1, 2, 4, 8, and 16 µg/mL) in a liquid growth medium dispensed in test tubes » Determines the MIC of a drug that inhibits growth of a particular organism in vitro  Culture Methods Nucleic Acid Based Methods Used to detect known resistance genes or mutations. E.g., mecA, a gene for oxacillin resistance in S. aureus Nucleic acid methods are used for: Rapid diagnosis of multidrug-resistant tuberculosis in at-risk groups Rapid detection of possible resistance in organisms directly obtained from positive blood cultures Culture Methods Automated methods MALDI-TOF MS Vitex 2 compact Microscan 3