Bacterial Cultures PDF

BACTERIAL CULTURES  MICROBIAL CULTURE A microbial culture is a method of multiplying microbial organisms (bacteria and fungi) by letting them reproduce under controlled laboratory conditions. The in vitro cultures of bacteria allow: → isolation and cultivation of th...

BACTERIAL CULTURES  MICROBIAL CULTURE A microbial culture is a method of multiplying microbial organisms (bacteria and fungi) by letting them reproduce under controlled laboratory conditions. The in vitro cultures of bacteria allow: → isolation and cultivation of the pathogenic bacteria → information on the morphology of the colonies, phenotypic characteristics necessary for bacterial identification → information on the antibacterial susceptibility of the pathogenic bacteria for selective antimicrobial agents MICROBIAL CULTURE PLAYS A SIGNIFICANT ROLE IN IDENTIFYING THE CAUSATIVE AGENT OF A PARTICULAR DISEASE  CULTURE MEDIA Culture media are nutrient media or preparations that support and allow microorganisms to be propagated in the laboratory for further study. They are artificial growth medium. Culture media required for bacteriological invetigations are basically classified into 3 categories according to: o Consistency (physical state): solid, semi-solid, liquid or broth o Nutritional components: complex or synthetic media ✓ Complex culture media (or undefined) contain unknown amount of some nutrients, and they are mainly used for the cultivation of fastidious bacteria (require additional nutrients as blood, serum, yeast, meat extract whose exact chemical makeup is uncertain) ✓ Synthetic culture media (or chemically defined) contain only known and well-defined chemical constituents o Function or property: include selective media, differential or indicator media, enriched media, composite media, storage media, transport media LIQUID CULTURE MEDIA TURBIDITY  SOLID CULTURE MEDIA agar solidifying agent made up of long chain of D- galactose, obtained from red-purple marine algae Agar is unique, and an incomparable ingredient of solid media because of its solidifying properties. Agar is: o physiologically stable upon usage and storage o nontoxic to bacterial growth o stable during sterilization o not degraded by most pathogenic bacteria Agar liquifies at 100°C and solidifies when it is cooled to 50°C SOLID MEDIUM FOR BACTERIA GROWTH CURVE FOR A BACTERIAL POPULATION  CONTINUOUS CULTURE DEVICE: CHEMOSTAT - BIOREACTOR Chemostat system with a continuous inflow of fresh medium/air and outflow of effluent for controlled growth of microorganisms The chemostat allows researchers to maintain the Log growth phase of a bacterial population BACTERIAL GROWTH Vs OXYGEN CONCENTRATION  BACTERIAL GROWTH IN SOLID MEDIA Bacterial growth in solid culture is observed as colony formation. Bacteria form isolated colonies starting from growth centers formed by single cells.  TYPES OF CULTURE MEDIA Enriched nonselective media o Blood agar contains 5% sheep/horse blood. It tests bacteria’s ability to lyse red blood cells. Lysis of erythrocytes is called hemolysis and is catalyzed by enzymes called hemolysins. o Chocolate agar is a modified blood agar obtained by heating the blood until it turns chocolate-brown in color.  TYPES OF CULTURE MEDIA Enriched nonselective media o Mueller-Hinton agar is the recommended medium for antibiotic susceptibility testing Disk diffusion test, or agar diffusion test, or Kirby–Bauer test  DISC DIFFUSION TEST Ø 6 mm Loaded discs with antimicrobials Sterile discs to be impregnated with 10 µL of sample  DISC DIFFUSION TEST The Kirby–Bauer test or disk diffusion test, or agar diffusion test, is an antimicrobial susceptibility test performed on Mueller-Hinton agar plate. It uses discs to test the extent to which microbes are affected by those drugs. In this test, paper discs containing the drugs are placed on an agar plate where microbes have been placed, and the plate is left to incubate. If the drug stops the microbes from growing or kills the microbes, there will be an area around the disc where the microbes have not grown enough to be visible. This is called a zone of inhibition. Paper disk Zone of inhibition Bacterial growth ANTIBIOTIC SUSCEPTIBILITY TESTING IN LIQUID MEDIUM MINIMAL INHIBITORY CONCENTRATION (MIC) ANTIBIOTIC SUSCEPTIBILITY TESTING IN LIQUID MEDIUM MINIMAL INHIBITORY CONCENTRATION (MIC)  TYPES OF CULTURE MEDIA Selective media and differential media Selective media are designed for the recovery of specific bacteria that may be present in a mixture of other microorganisms (enteric pathogens in fecal specimens) → they are supplemented with inhibitors that suppress the growth of unwanted organisms. Selective media can be made differential by adding specific substances that allow identification of an organism in a mixture (example: addition of lactose and a pH indicator to detect lactose fermenting bacteria). Some examples of selective and differential media: Mannitol salt agar Lowestein-Jensen agar CHROMagar  TYPES OF CULTURE MEDIA Lowestein-Jensen Selective for the isolation of medium mycobacteria. It contains glycerol, potato flour, salts, and coagulated whole eggs (to solidify the medium). Malachite green is added to inhibit gram-positive bacteria  TYPES OF CULTURE MEDIA CHROMagar Orientation