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biotopics.co.uk/microbes/tech1.html

http://www.biotopics.co.uk/microbes/tech1.html. Bacteria Requirements. Need an organic source of Carbon to respire for energy & a source of Nitrogen to incorporate into proteins In nature : substances are digested with enzymes (extracellular digestion) & nutrients are absorbed across cell wall

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biotopics.co.uk/microbes/tech1.html

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  1. http://www.biotopics.co.uk/microbes/tech1.html

  2. Bacteria Requirements • Need an organic source of Carbon to respire for energy & a source of Nitrogen to incorporate into proteins • In nature: substances are digested with enzymes (extracellular digestion) & nutrients are absorbed across cell wall • In lab: we can dissolve substances into water • May also add vitamins, minerals, pH buffers, antibiotics… depends on the bacteria you’re trying to grow Antibiotics prevent the bacteria from growing as can be seen in the clear area

  3. Types of Growth Medium • Nutrient broth: clear soup-like liquid usually in tubes • Nutrient agar: nutrients mixed with hot-liquid agarose (seaweed extract) which will solidify into a Jell-O type of consistency • Petri-dishes (plates) • Petri-Film

  4. Sterile Technique • All equipment is sterilized to ensure that any existing bacteria are killed • Media: sterilize with an autoclave (uses heat & pressure to kill bacteria) • Equipment: sterilize with heat (ex: from a Bunsen Burner), minimize exposure of media to the air

  5. Safety • Always wash your hands & your lab area both before & after lab • Anything you grow is kept sealed unless instructed otherwise • To dispose of cultured (grown) bacteria: • Bleach water • Autoclave

  6. Introducing Bacteria to the Media (inoculation) • Usually: use an inoculation loop (sterile) • Sometimes: sterile cotton swab • Sometimes: bacteria in a liquid are introduced using a sterile pipette to the Petri dish before the (fairly cool) agar medium is poured on top ("pour plates")

  7. Incubation • Incubator: special apparatus set to a specific temperature (ex: 37ºC) for 24-48 hours • For petri dishes: invert the dish so that condensation accumulates on the lid, away from the bacteria • For Petri Film: can be stacked on top of each other (up to 20 films)

  8. Why use a particular growth medium?

  9. NUTRIENT BROTH Flame the opening of tube to sterilize! Nutrient broth turns cloudy when bacteria grow during incubation

  10. PETRI DISH (PLATE) Colony counter: -current models attempt to identify individual areas of dark and light according to automatic or user-set thresholds, and then count the resulting contrasting spots. *Maximum # can count is between 100-1000 depending on organism -old models put the plate on a lighted platform & you marked off each colony with a Sharpie Bacterial colonies: “pile” of identical bacteria that arose from 1 original bacteria through binary fission Fuzzy = fungus YUCK!

  11. Streaking helps you isolate colonies of bacteria; in the 1st steaks, those bacteria cannot be quantified Spreading sample like this, would allow colonies to be quantified as long as there isn’t a lawn

  12. Dense and overlapping E. coli colonies Colonies merge into a single lawn. Notice that the lawn is translucent, in contrast to the nearly transparent agar.  The edge of the lawn is visible near the edge of the plate. *If you think you are sampling something that you would expect to have a large # of bacteria, then you need to discuss how to dilute your sample in order to be able to quantify the # of colonies.*

  13. PETRI FILM Think to yourself: How could you estimate this high number of bacteria? Petri spreader: How did you think they got such perfect circles?

  14. Microbiology DESIGN Lab Topic: Comparing bacterial growth on surfaces found at NPHS. • Questions to consider when forming your question: • Do you want to choose different surfaces or 1 surface at different times? • Why would anyone be interested in finding out about how much bacteria there is on that surface?

  15. Questions to consider as you design a procedure: • How can you ensure your safety & the safety of others before/while collecting & growing bacteria? • How are you going to ensure you don’t have any bacterial contamination? • Have you included detailed steps of how to get your sample? • This is where controls are going to be important! • Think of this as if you are telling someone to do what you ask over the phone since you can’t do it yourself. • What growth medium are you going to use? • Where are your bacteria growing? • How are you going to collect data? • Does your procedure describe what to do if you have an overabundance of bacteria (a lawn)? • How do you ensure everyone's safety after bacteria have been grown?

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