M 2 culture methods by Dr.vimal prakash
- 2. PURPOSE •Pure culture •Biochemical tests •Antimicrobial susceptibility testing •Stock culture •Sufficient growth-antigens •Typing •Viable bacterial counts
- 3. METHODS Loops and Straight wires: •Bacteriogical loops-2to4mm-Streaking •Bacteriological Straight wires-Stroke and Stab culture •Bunsen flame-redhot-cool-10secs •Biological safety cabinet
- 8. STREAK CULTURE •Isolation •Individual isolated –mixed culture •Method of streaking: •Incubation 37deg C -12to18hrs •Biochemical tests
- 11. LAWN OR CARPET CULTURE •Swabbing •Flooding USES: •AST •Bacteriophage typing •Bacterial grth-Preparation of antigens and vaccines
- 13. STROKE CULTURE Agar slopes or slants-zigzag fashion •Diagnostic tests •Citrate utilization test and urease test
- 15. STAB CULTURE Stabbing semisolid agar butt-straight wire •Stock culture •OF test •Motility testing •Eg. MMM , Nutrient agar semisolid butts , TSI.
- 17. LIQUID CULTURE Touching with a loop or Adding the with pipettes or Syringes Bacterial growth: •Uniform turbidity •Granular turbidity •Surface pellicles Uses: •Blood culture •Sterility testing •Water analysis
- 19. POUR PLATE CULTURE Viable bacterial count •Number of bacteria per ml of liquid broth/specimen ▪Serial 10 fold dilutions ▪Pour plating ▪Colony counting ▪Viable count per ml
- 22. SPREAD-PLATE METHOD Known volume of individual dilutions are spread evenly on the surface of agar plate to obtain lawn culture After incubation colonies are counted &multiplied by DF to estimate bacterial count
- 23. INCUBATION OF CULTURE MEDIA •Pathogenic organisms-37 C •Aerobic bacteria-culture plates – 37 C •Overnight in an incubator •Bacteriological incubator
- 25. INCUBATORY CONDITIONS •Candle jar Capnophilic bacteria-Brucella abortus, Streptococcus, Pneumococcus,Gonococcus •Microaerophilic bacteria-Campylobacter, Helicobacter Require 5% oxygen -growth
- 27. ANAEROBIC CULTURE METHODS Obligate anaerobes •Production of vacuum-vacuum desicator •By displacement and combustion of oxygen MCINTOSH AND FILDES ANAEROBIC JAR: Evacuation of air from jar and replacement with inert gas like hydrogen followed by removal of residual oxygen by catalyst. Aluminium pellets coated with palladium Removal of residual oxygen (O2+H2-------H2O) ANOXOMAT: Evacuation of air from the jar and replaces by hydrogen gas from a cylinder.Same catalyst. Effective method.
- 30. •ABSORPTION OF OXYGEN BY CHEMICAL METHODS Chemical reactions GASPAK SYSTEM Sachet-sodium bicarbonate andsodium borohydride Chemical indicator-reduced methylene blue Biological indicator –Pseudomonas •ANAEROBIC GLOVE BOX(ANAEROBIC CHAMBER) Without O2 •BY USING VARIOUS REDUCING AGENTS Glucose,thioglycollate,cooked meat pieces, Robertson cooked meat broth-chopped meat •PREREDUCED ANAEROBICALLY STERILIZED(PRAS) Sealed foiled pockets
- 33. PRESERVATION OF MICROORGANISMS Epidemiological investigation,future research and educational purposes. •Short term-weeks to months •Long term-years
- 34. SHORT TERM METHODS Phenotypic and genotypic-altered •Subculture-cookedmeat medium-anaerobes •Mineral oil,glycerol,sterile distilled water •Freezing at -20 deg C •Drying-moulds,spore bearing bacteria
- 35. LONG TERM PRESERVATION METHODS Advantages Less space,phenotypic and genotypic maintained,reduced chance of mutation,viability well maintained. •Ultra temperature freezing-cryopreservative agents- glycerol,skimmed milk,sucrose and incubation at -70deg C •Lyophilization-freezing of liquid culture followed by dehydration to remove water from frozen bacterial suspensions Stable readily rehydrated product-4deg C
- 36. METHODS OF ISOLATING BACTERIA IN PURE CULTURE ▪Surface plating-Intermittentheat ▪Selective media and enrichment broth-eg faeces ▪Pretreatment of specimens-4% ▪Anaerobiasis ▪Heating-thermophilic 60 deg c ▪Filters-different pore diam-sizes ▪Based on motility-SC in craigie ▪Animal inoculation-guinea pigs
- 37. THANK YOU