BACTERIAL CULTURE                    METHODS                                     BASICS                     Dr.T.V.Rao MDD...
BACTERIAL NUTRITION AND THE DESIGN           OF CULTURE MEDIA• Based on bacterial  metabolism*• Culture pH• Culture oxidat...
CULTURE MEDIA• Used to grow bacteria• Can be used to:      • Enrich the numbers of        bacteria      • Select for certa...
OXYGEN CONCENTRATION• Aerobs• Anaerobs (do not require  oxygen)• Obligate anaerobs (die in  the presence of Ooxygen  )• Fa...
PURPOSE OF CULTURING• Isolation• Properties of bacteria• To create antigens for  laboratory use• Typing with Bacteriophage...
METHODS OF ISOLATION OF PURE         CULTURE WITH ..• 1. Surface plating• 2 Enrichment  medium• 3 Selective medium• 4 Indi...
TYPES OF MEDIA USEDGeneral purpose media will  support the growth of many  microorganisms.Enriched media are general  purp...
TYPES OF MEDIA USEDSelective media favor the   growth of particular   microorganisms and inhibits   the growth of others.D...
TEMPERATURE ( CHARACTERISTIC RANGES)• Psychrophiles: with optimum  growth T around 20 C• Mesopihles: between 15 and  45 wi...
TEMPERATURE AND BACTERIAL              GROWTHDR.T.V.RAO MD                     10
DR.T.V.RAO MD   11
THE REQUIREMENTS FOR GROWTH:         PHYSICAL REQUIREMENTS• pH      • Most bacteria grow        between pH 6.5 and        ...
CULTURING• Used to grow bacteria• Can be used to:     • Enrich the numbers of       bacteria     • Select for certain bact...
METHODS TO ISOLATE THE                      BACTERIA• Streak culture• Stroke• Stab• Pour plate• Liquid culture• Special me...
How to inoculate a plate     HOW TO INOCULATE A CULTURE PLATE• Plate: provide large  surface for isolation and  observatio...
DIFFERENT METHODS OF CULTURING              BACTERIADR.T.V.RAO MD                       16
MACCONKEY AGAR• Example:: MacConkey agar  has color indicator that  distinguishes presence of  acid. Bacteria that ferment...
COLONIES - MAKE A OBSERVATION                      • Shape                      • Size                      • Elevation   ...
LIQUID MEDIALiquid media: easiest to  prepare and use. Good for  growing quantities of  microbes needed for  analysis or e...
SLANT OBSERVATION                http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/slant_patterns.jpgDR.T.V.RAO M...
STREAK CULTURE                       • Lawn or carpet culture                         to create uniform                   ...
CULTURING THE MICROBES NEEDS SKILLSDR.T.V.RAO MD                      22
STREAK PLATEDR.T.V.RAO MD                        23                               Figure 6.10a–b
METHODS OF ISOLATION OF PURE                 CULTURE• 1. Surface plating• 2 Enrichment  medium• 3 Selective medium• 4 Indi...
LIQUID CULTURING• Liquid cultures are done  in• Tubes• Bottles• Flasks• Blood culture• Water analysis DR.T.V.RAO MD       ...
STAB CULTURE                      • Puncturing suitable                        medium such as                        nutri...
SWEEP PLATE METHODDR.T.V.RAO MD                    27
MICROBIAL ANTIBIOTIC SUSCEPTIBILITY               TEST   (THE AGAR DIFFUSION TEST )                            Zones of   ...
MULLER HINTON AGAR FOR ANTIBIOTIC TESTINGDR.T.V.RAO MD                           29
MEASURING THE ZONE OF                     INHIBITIONDR.T.V.RAO MD                           30
MINIMUM INHIBITORY CONCENTRATION           DETECTS ANTIBIOTIC SENSITIVITY PATTERNSDR.T.V.RAO MD                           ...
• Anaerobic Bacterial Isolation    and Identification Needs      specified conditionsDR.T.V.RAO MD                 32
DESICCATOR• In Desiccator some oxygen  is left  Not suitable for fluid cultureDisplacement of oxygen is  done with  Hydrog...
CANDLE JAR• Inoculated plates are kept• Burning candle use up all  oxygen• But a little o2 is left• But presence of Co2  s...
MAC IN TOSH FILDES ANAEROBIC JAR• Contain inlet and outlet• Electrical supply• Inoculated culture plates• When electrified...
GAS PACK                    • A disposable envelop contains                      chemicals which generate hydrogen        ...
OTHER REDUCING AGENTS• Reducing agents O.1% Thiglyclolate 0.1% Ascorbic acid 0.05 % cysteineDR.T.V.RAO MD                 ...
ROBERTSON COOKED MEAT MEDIUM ROUTINELY USED    IN ANAEROBIC SPORE BEARING BACTERIADR.T.V.RAO MD                           ...
DR.T.V.RAO MD   39
LOWENSTEIN JENSEN MEDIUM - CULTIVATION OF            MYCOBACTERIUM TUBERCULOSISDR.T.V.RAO MD                              ...
WORKING WITH MYCOBACTERIUM NEEDS         BIOSAFTEY CONCERNSDR.T.V.RAO MD                         41
• Created by Dr.T.V.Rao MD for Basic learning        on Culturing Bacteria for Medical and        Paramedical students in ...
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  1. 1. BACTERIAL CULTURE METHODS BASICS Dr.T.V.Rao MDDR.T.V.RAO MD 1
  2. 2. BACTERIAL NUTRITION AND THE DESIGN OF CULTURE MEDIA• Based on bacterial metabolism*• Culture pH• Culture oxidation- reduction potential.• Gaseous requirements • Oxygen, Carbon dioxide and other gasesDR.T.V.RAO MD 2
  3. 3. CULTURE MEDIA• Used to grow bacteria• Can be used to: • Enrich the numbers of bacteria • Select for certain bacteria and suppress others • Differentiate among different kinds of bacteriaDR.T.V.RAO MD 3
  4. 4. OXYGEN CONCENTRATION• Aerobs• Anaerobs (do not require oxygen)• Obligate anaerobs (die in the presence of Ooxygen )• Facultative anaerobs (E.coli)• Microaerophilic bacteriaDR.T.V.RAO MD 4
  5. 5. PURPOSE OF CULTURING• Isolation• Properties of bacteria• To create antigens for laboratory use• Typing with Bacteriophages and Bacteriocins susceptibility• To test for Antibiotic sensitivity• Estimate viable counts• Maintain stock culturesDR.T.V.RAO MD 5
  6. 6. METHODS OF ISOLATION OF PURE CULTURE WITH ..• 1. Surface plating• 2 Enrichment medium• 3 Selective medium• 4 Indicator mediumDR.T.V.RAO MD 6
  7. 7. TYPES OF MEDIA USEDGeneral purpose media will support the growth of many microorganisms.Enriched media are general purpose media supplemented by blood or other special nutrients to encourage the growth of fastidious heterotrophs; (fastidious = having complicated nutritional requirementsDR.T.V.RAO MD 7
  8. 8. TYPES OF MEDIA USEDSelective media favor the growth of particular microorganisms and inhibits the growth of others.Differential media distinguish between different groups of bacteria on the basis of their biological characteristics; Causes observable change in medium when biochemical reaction occursDR.T.V.RAO MD 8
  9. 9. TEMPERATURE ( CHARACTERISTIC RANGES)• Psychrophiles: with optimum growth T around 20 C• Mesopihles: between 15 and 45 with optimum around 37 C• Thermophiles: between 30 and 75 with optimum around 55 C• Hyperthermophiles: T grater than 100CDR.T.V.RAO MD 9
  10. 10. TEMPERATURE AND BACTERIAL GROWTHDR.T.V.RAO MD 10
  11. 11. DR.T.V.RAO MD 11
  12. 12. THE REQUIREMENTS FOR GROWTH: PHYSICAL REQUIREMENTS• pH • Most bacteria grow between pH 6.5 and 7.5 • Molds and yeasts grow between pH 5 and 6 • Acidophilic grow in acidic environmentsDR.T.V.RAO MD 12
  13. 13. CULTURING• Used to grow bacteria• Can be used to: • Enrich the numbers of bacteria • Select for certain bacteria and suppress others • Differentiate among different kinds of bacteria DR.T.V.RAO MD 13
  14. 14. METHODS TO ISOLATE THE BACTERIA• Streak culture• Stroke• Stab• Pour plate• Liquid culture• Special methods for anaerobic culturesDR.T.V.RAO MD 14
  15. 15. How to inoculate a plate HOW TO INOCULATE A CULTURE PLATE• Plate: provide large surface for isolation and observation of colonies• Using a sterile loop or a sterile swab streak your sample on the Petri plate• Important let your sterilized loop cool before you pick up your sample DR.T.V.RAO MD 15
  16. 16. DIFFERENT METHODS OF CULTURING BACTERIADR.T.V.RAO MD 16
  17. 17. MACCONKEY AGAR• Example:: MacConkey agar has color indicator that distinguishes presence of acid. Bacteria that ferment a particular sugar (e.g., glucose in culture media) will produce acid wastes on plates, turn pH indicator red. DR.T.V.RAO MD 17
  18. 18. COLONIES - MAKE A OBSERVATION • Shape • Size • Elevation • Edge • Surface • Opacity • ConsistencyDR.T.V.RAO MD 18
  19. 19. LIQUID MEDIALiquid media: easiest to prepare and use. Good for growing quantities of microbes needed for analysis or experiments. Unless inoculated with pure culture, cannot separate different organisms. DR.T.V.RAO MD 19
  20. 20. SLANT OBSERVATION http://www.rlc.dcccd.edu/MATHSCI/reynolds/MICRO/lab_manual/slant_patterns.jpgDR.T.V.RAO MD 20
  21. 21. STREAK CULTURE • Lawn or carpet culture to create uniform surface of organisms • Bacteriophages typing • To obtain large amount of antigensDR.T.V.RAO MD 21
  22. 22. CULTURING THE MICROBES NEEDS SKILLSDR.T.V.RAO MD 22
  23. 23. STREAK PLATEDR.T.V.RAO MD 23 Figure 6.10a–b
  24. 24. METHODS OF ISOLATION OF PURE CULTURE• 1. Surface plating• 2 Enrichment medium• 3 Selective medium• 4 Indicator mediumDR.T.V.RAO MD 24
  25. 25. LIQUID CULTURING• Liquid cultures are done in• Tubes• Bottles• Flasks• Blood culture• Water analysis DR.T.V.RAO MD 25
  26. 26. STAB CULTURE • Puncturing suitable medium such as nutrient agar, gelatin, • Observe gelatin liquefaction • Preserving the stock culture.DR.T.V.RAO MD 26
  27. 27. SWEEP PLATE METHODDR.T.V.RAO MD 27
  28. 28. MICROBIAL ANTIBIOTIC SUSCEPTIBILITY TEST (THE AGAR DIFFUSION TEST ) Zones of growth inhibitionDR.T.V.RAO MD 28
  29. 29. MULLER HINTON AGAR FOR ANTIBIOTIC TESTINGDR.T.V.RAO MD 29
  30. 30. MEASURING THE ZONE OF INHIBITIONDR.T.V.RAO MD 30
  31. 31. MINIMUM INHIBITORY CONCENTRATION DETECTS ANTIBIOTIC SENSITIVITY PATTERNSDR.T.V.RAO MD 31
  32. 32. • Anaerobic Bacterial Isolation and Identification Needs specified conditionsDR.T.V.RAO MD 32
  33. 33. DESICCATOR• In Desiccator some oxygen is left Not suitable for fluid cultureDisplacement of oxygen is done with Hydrogen Nitrogen Helium Co2 DR.T.V.RAO MD 33
  34. 34. CANDLE JAR• Inoculated plates are kept• Burning candle use up all oxygen• But a little o2 is left• But presence of Co2 stimulates the most bacterium DR.T.V.RAO MD 34
  35. 35. MAC IN TOSH FILDES ANAEROBIC JAR• Contain inlet and outlet• Electrical supply• Inoculated culture plates• When electrified palladinised asbestos heating acts as catalyst for combination of hydrogen with residual oxygen causes complete anaerobiasis DR.T.V.RAO MD 35
  36. 36. GAS PACK • A disposable envelop contains chemicals which generate hydrogen and carbon dioxide on addition of water • Inoculated plates are kept in jar • Water is added hydrogen and carbon dioxide are liberated • Presence of cold catalyst in the envelop permits the combination of Hydrogen and oxygen to produce anaerobic environment • Indicator is methylene blue • Colorless when anaerobic environment.DR.T.V.RAO MD 36
  37. 37. OTHER REDUCING AGENTS• Reducing agents O.1% Thiglyclolate 0.1% Ascorbic acid 0.05 % cysteineDR.T.V.RAO MD 37
  38. 38. ROBERTSON COOKED MEAT MEDIUM ROUTINELY USED IN ANAEROBIC SPORE BEARING BACTERIADR.T.V.RAO MD 38
  39. 39. DR.T.V.RAO MD 39
  40. 40. LOWENSTEIN JENSEN MEDIUM - CULTIVATION OF MYCOBACTERIUM TUBERCULOSISDR.T.V.RAO MD 40
  41. 41. WORKING WITH MYCOBACTERIUM NEEDS BIOSAFTEY CONCERNSDR.T.V.RAO MD 41
  42. 42. • Created by Dr.T.V.Rao MD for Basic learning on Culturing Bacteria for Medical and Paramedical students in Microbiology • Email • doctortvrao@gmail.comDR.T.V.RAO MD 42
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