Recommended
More Related Content
What's hot
What's hot (20)
Similar to Micro Lab 3 Lecture
Similar to Micro Lab 3 Lecture (20)
More from stanbridge
More from stanbridge (20)
Recently uploaded
Recently uploaded (20)
Micro Lab 3 Lecture
- 2. The Five “I’s 1. Inoculation: Method of transferring organisms used to produce a pure culture 2. Isolation: Selection of a single type of colony on media (CFU) with one single kind of microbe growing (pure culture/isolated colony) 3. Incubation: Method of growing microbes under proper conditions 4. Inspection: Observation of characteristics, data and results 5. Identification: use of data, comparison correlation, to ID organism to exact species
- 3. 1. Inoculation: Producing a pure cultures • Colony – macroscopically visible collection of bacteria originating from a single bacterial cell. • Colony Forming Units (CFU) – All the genetically related progeny of a single cultured organism or cell growing as colony. • Pure cultures – a single organism and its reproduced progeny
- 4. 1. Inoculation: • Introduce bacteria into a growth medium using “aseptic technique” to prevent contamination. • Tools: Bunsen burner, loop. Needle, etc.
- 5. Inoculation: Agar Gel • Frau Hesse • Used for preparing solid medium • Obtained from seaweeds. • No nutritive value • Not affected by the growth of the bacteria. • Melts at 98oC & sets at 42oC • 2% agar is employed in solid medium
- 6. 2. Isolation: Producing CFU • Isolation: – Growth media/medium: • Liquid/gel designed to support the growth of microbes, cells or small plants – General : • NA (nutrient agar), TSA (Trypticase soy agar) – Differential: Have dyes, salts, inhibiting agents • Mac (selectively isolate Gram- ve & enteric bacilli and differentiate them based on lactose fermentation. • EMB (Eosin methylene blue is a selective stain for gram-negative bacteria) • SS (Salmonella-Shigella , is a selective and differential medium . It is used for the isolation, cultivation and differentiation of gram-negative
- 7. Pure Cultures • Pure culture: culture of bacteria in which the organisms are all genetically identical – All of the organisms are the same species and contain the exact same DNA (clones) – Usually they are all derived from the same original cell • Mixed Culture – culture of bacteria in which the organisms are not genetically identical – The organisms are different species and/or contain different DNA
- 8. • Pure cultures are necessary to ensure the integrity of your results – Different bacteria have different properties and respond differently to chemicals and other manipulations – If your culture is not pure, how can you be sure your results are accurate? • Contamination by another type of microorganism can ruin all of your work Why are pure cultures important?
- 9. What is an “isolated colony?” • Colony – aka Colony Forming Unit (CFU) – Small culture of bacteria, on solid media • all derived from the same original bacterial cell – They are all genetically identical (therefore, this is a type of pure culture) • Isolated colony – CFU not touching or questionably close to any other CFU on the plate or slant – Obtaining isolated CFUs from a culture (mixed or pure) is the point of the 3 techniques
- 10. Types of culture media 1. Based on consistency a) solid medium b) liquid medium c) semi solid medium
- 11. Solid media – contains 2% agar • Colony morphology, pigmentation, hemolysis can be observed. • Eg: Nutrient agar, Blood agar Liquid media – no agar. • For inoculum preparation, Blood culture, for the enrichment of pathogens from a mixture. • Eg: Nutrient broth, Selenite broth Semi solid medium – 0.5% agar. • Eg: Motility medium
- 12. Types of culture media II. Based on the constituents/ ingredients a) simple medium b) complex medium c) synthetic or defined medium d) Special media Special types of media: – Enriched - Enrichment – Selective - Indicator - Differential – Sugar - Transport - Biochemical reactions
- 13. Simple media / basal media • Eg: Nutrient Broth, Nutrient Agar, Tryptic Soy Agar • NB consists of peptone, meat extract, NaCl, • NB + 2% agar = Nutrient Agar
- 14. Complex media • Media other than basal media • They have added ingredients • Provide special nutrients Synthetic or defined media • Media prepared from pure chemical substances and its exact composition is known • Eg: peptone water – 1% peptone + 0.5% NaCl in water
- 15. Enriched media • Substances are added to the basal medium – blood, serum, egg – For bacteria with strict nutritional needs • Blood agar (L) • Chocolate agar (R)
- 16. Enrichment media • Liquid media used to enrich pathogens in mixed cultures • Media is incorporated with inhibitory substances to suppress unwanted organisms (e.g normal flora) • Selenite F Broth – Isolation of Salmonella, Shigella • Alkaline Peptone Water – Isolation of Vibrio cholerae
- 17. Selective media • Inhibitory substances added to solid media • MacConkey – For gram -ve bacteria – Has crystal violet and bile salts to inhibit gram +ve organisms – Lactose fermentation is differential • TCBS (Thiosulfate-citrate-bile salts-sucrose ) – For Vibrios – Has sodium thiosulfate and sodium citrate to inhibit Enterobacteriaceae – Sorbitol fermentation is differential
- 18. Selective media • LJ (Lowenstein Jensen) medium – M. tuberculosis – Malachite green, eggs, etc • Potassium tellurite medium – Diphtheria bacilli
- 19. Indicator media • Contain an indicator which changes its color when a bacterium grows in them. – Blood agar – MacConkey – Christensen’s urea
- 20. Blood agar • Rich with nutrients • Differential (can see hemolysis)
- 22. Urease medium
- 23. Differential: • Mac, EMB, SS • These have dyes, salts, inhibiting agents – Visual differences on plates
- 24. Differential media • Has substances which permit differentiation. – Mac Conkey’s medium – Peptone – Lactose – Agar – Neutral red – Taurocholate (bile salts) • Distinguish between: – Lactose fermenters • Pink colonies (E. coli) – Non lactose fermenters • Colorless (Salmonella)
- 25. Sugar media • Media containing fermentable substance – Glucose – Arabinose – Lactose – Starch etc • Contains 1% sugar, peptone, phenol red • Durham tube – small tube for gas detection
- 26. III.Based on Oxygen requirement - Aerobic media - Anaerobic media Types of culture media
- 27. Isolation: Producing CFU • Plating methods
- 28. Isolation: Streak Plates: not suitable for plate counts
- 29. Smear Plate Rotation of glass wand spread microbes across entire plate
- 31. 3. Incubation: • Allow organisms to grow under the optimal conditions • Temperature regulation • With or without oxygen • CO2%, etc.
- 32. Incubation: • Allow organisms to grow under the optimal conditions • Temperature, with or without oxygen etc. • Candle jar reduces oxygen
- 33. 4. Inspection: • Observation, description • Colony Morphology, Microscopic examination (grams stain) • Systematic recording of “DATA”
- 34. Inspection Gram (+) bacilli Gram (-) bacilli
- 35. Acid fast, and capsule
- 36. 5. Identification: • Correlating data from all observations and resources to ID organism to species – Flow charts – Bergey’s manual, etc. – API (Analytical profile index)
- 37. 5. Identification: • Correlating data/resources to ID organism – Gram (+) – Cocci – “Grape” clusters – Golden yellow colonies, – Catalase + – Coagulase + – Resistant to Methicillin (MRSA, Methicillin-resistant Staphylococcus aureus ) • Staphylococcus aureus
- 39. Today’s Lab
- 40. HOW TO OBTAIN PURE CULTURES: Methods of Bacterial Separation Quadrant Streak Plate: • Method to dilute inoculum and obtain well- isolated and discrete colony forming units • Spread Plate: Method of amounts of organisms often for the purpose of counting, using a glass rod
- 41. Today • Describe 6 plates – Two on simple media (TSA) – Two on enriched media (BAP) – Two on selective media (MAC) – Note: form, elevation, margin, color, hemolysis (BAP only), lactose reaction (MAC only)
- 42. Today • Prepare 3 Streak plates – One on simple media – One on enriched media – One on selective media