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Microscopy and Bacteria Observation Lab Activities

Develop knowledge of microscopy and microbial observation techniques. Learn to discern and identify different bacteria types. Practice aseptic techniques and isolation methods for pure cultures. Understand bacterial contamination and growth media.

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Microscopy and Bacteria Observation Lab Activities

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  1. Exercise 2: 2A: Microscopy

  2. Announcements • Post Lab 2 is assigned today and due by the time your lab meets next. • Pre Lab 3 will be available on Wednesday at 5 PM and is also due by the time your lab meets next. • LNA Bacteria is assigned today, and due by the time your lab meets next*. • Pre-Lab Write for LNA 3 is due within the first 5 minutes of lab next week.

  3. Goals • Develop working knowledge of a brightfield light microscope • Discern between different types of microscopy • Practice techniques: objectives, oil, wet mount, measurements

  4. Practice • Practice with the objectives, focusing, and positioning using the prepared slides. • Use your lab manual as a reference if you are having trouble pages 29-30.

  5. Bacterial Observation . .

  6. Observation of Bacteria • Sphere • cocci • Rod • bacilli • Spiral • Spirochete and spirilla

  7. Exercise 2: 2B: Bacteria

  8. Goals • Become familiar with the scientific process by generating hypotheses, making predictions and designing experiments • Determine potential sources of microbial contamination in the laboratory • Obtain a pure culture of bacteria by streaking for isolated colonies on solid media

  9. Bacteria • Prokaryotes: lack a nuclear membrane • Small, single cell organisms • Exist in huge numbers in small amounts of material • Found almost everywhere

  10. Contamination • Look for contamination by testing for growth on bacterial medium.

  11. Pure Cultures • A population of cells, all of which are descended from a single cell.

  12. Growth Media • Liquid or Solid • Agar • Non-toxic • Remains solid at high temperatures • Not used as a nutrient • Defined or Complex

  13. Sterilization • To kill all living organisms • Autoclaving • Baking • Alcohol • Flaming • Filtration

  14. Aseptic Technique • Inoculating Loop • Serological Pipettes • Microliter Pipettes

  15. Colony • Cluster of cells visible to the naked eye • Facilitating: • Isolation of pure cultures • Enumeration of cell concentration in liquid suspensions • ID of bacterial species based upon the appearance of the colonies

  16. Streaking Plates .

  17. Spreading Plates • A diluted suspension is pipetted directly onto the surface of an agar plate and spread across the surface using a sterile glass spreader.

  18. Determining the number of viable bacteria • The number of colonies on a plate is assumed to be equal to the number of viable cells which were spread on the plate • Limiting Factors • No more than 0.2 ml of cell suspension should be spread on the plate • Resulting in 30 to 300 cells spread on the plate

  19. Determining Cell Mass by Measuring Turbidity • The number of cells per ml is directly proportional to the mass of cells per ml • Using Spectrophotometers to measure Optical Density (OD) • Light is lost as it passes through the suspension because it is scattered and absorbed by the cells. • Data can be used to construct a standard curve.

  20. A Standard Curve of Viable Cells Versus Turbidity

  21. Bacterial Stains • Positive Stains: cells pick up color • Negative Stains: background color, cells appear white

  22. Procedure Part I • Labeling plates • Label the bottom of the agar plate • Write on the periphery of the plate • Write your name, section, date, and location • Use the sterile swab to collect your contaminant and put it on the agar plate • Incubate plates for 48 hours at 37ºC

  23. Procedure Part II • Isolate Pure Cultures • Using Aseptic Technique • E. coli • Use Streak Techniques

  24. First LNA: Due Next Lab • You should come to lab next with with your lab notebook assignment. • There will be enough time at the beginning of next lab to observe your plates and finish the results and conclusions. • Also remember to have your Pre-Lab write-up (purpose, procedure, data table) for Exercise 3: Enzymes at the beginning of class.

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