Lecture 2Learning Objectives1. Name the different modes of light interaction with matter a. Transmission- Light goes through without being alteredb. Absorption- Object absorbs part of the light. Energy level raised in objectc. Reflection- Light bounces off the surfaced. Refraction- The light changes angles when it enters a substance that changes its phasee. Scattering- Some of the light is scattered in all directionsf. Emission- Some of the light is absorbed by the sample, which then produces light of a longer wavelength2. Explain which mode of light interaction with matter is used in each type of microscopea. Bright field Microscopei. The image results from:1. Light transmission through the sample2. Light absorption by some cell constituentsii. Poor contrastiii. Can only see size, shape and motileb. Oil-Immersion Objective: Oil makes light rays unrefracted i. Glass refractive index: 1.52ii. Immersion oil refractive index: 1.51iii. Air refractive index: 1.0iv. Benefit of using the oil-immersion objective: increases refractive index, yielding increased resolution c. Dark- field microscopes: Allow visualization of thin cells on a dark backgroundi. Some of the light reaching the specimen is scatteredii. Bright objects against black backgroundiii. Ideal for viewing very thin or small cells at the limit of light microscopy resolution = 0.2 umd. Phase contrast microscopy: Converts differences in refractive index within unstained cells into contrasti. Uses Light refraction 3. Explain which features of bacteria you can study by light microscopy; by fluorescence microscopya. Light i. Max resolution ~ 0.2umii. Dark field: very thin and small cellsiii. Phase contrast: Cell morphology of live cells; contrast b/t unstained bacteria and surroundingsb. Fluorescencei. Concept of fluorescence adapted to microscopyii. Fluorescent cells appear colored against a dark backgroundiii. Some microbes are auto fluorescent; some of their cell components (e.g.,chlorophyll) fluoresce, other microbes need to be stainediv. Can see the interaction between two organismsv. Interaction between microalga and growth promoting bacteriavi. Stain for specific DNA (DAPI)vii. Cyanobacteria, auto fluorescence and GFP fluorescenceviii. Live and Dead bacteria stained by BacLight4. Explain the mechanism of fluorescence 5. Identify which type of microscopy was used to obtain a given picture 6. Recall the different steps of the Gram stain a. Step 1: Flood the heat-fixed smear with crystal violet for 1 mini. Result: All cells purpleb. Step 2: Add iodine solution for 1 min, forms crystal violet-iodine complexi. Result: All cells remain purplec. Step 3: Decolorize with alcohol briefly- about 20 secondsi. Gram- positive cells are purple; gram negative cells are colorlessd. Step 4: Counterstain with safranin for 1-2 minutesi. Gram positive are purple; gram negative cells are pink to red7. Explain why Gram-negative and Gram-positive bacteria have different colors in the Gram stain. How do their membranes differ? a. Gram negative have 2 membranes and pepb. Gram positive have only one membrane and pep and the stain gets stuck in there and cant wash away so it stays purple8. Give examples of acid-fast bacteria a. Define: Differentiates mycobacteria form other bacteria. Mycobacteria cell waxes (= mycolic acid) specifically bind carbol fuchsin (pink) and retain when treated with acid- alcohol. Mycolic acid cannot be penetrated by gram stain.b. Examples: Mycobacterium tuberculosis, avium infection of lymph node in AIDS patient9. Explain why acid-fast bacteria cannot be stained with the Gram stain a. It retains the pink when treated with an acid alcohol so it will always look gram negative is gram stain is used10. Predict which color a certain bacterial species will be in the Gram or the acid-fast stain11. Compare and contrast TEM and SEM a. TEM: i. Thin sections of samples in resinii. Electrons go through the specimeniii. Contrast between transmitted and absorbed electron beams iv. Creates 2D images v. Negatively stainsb. SEMi. Image created by electrons reflecting off the specimen’s surfaceii. Creates 3D imageiii. Specimen surface coated with goldiv. Wide range of magnificationsv. High field depth (depth range in focus)12. List which features of bacteria you can study by TEM, or by SEM a. TEMi. Shows external coats, cell envelope and internal surfacesii. Negatively stain, whole cells or large molecules; shows cells surface texture and very thin external appendages iii. Cyanobacterial cell, thin sectioniv. Hemoglobin molecules, negative stainv. Bacterial cell with flagella, negative stainvi. Bacillus subtilis surrounded by 29 pages, thin sectionb. SEMi. Detects electrons that have been reflected off the object surfaceii. Specimen surface is coated with goldiii. Ideal for viewing surfaces colonized by bacteriaiv. Biofilm formed by E. coliv. Geobacter sulfurreducens cells attached to ferric iron precipitatesvi. Microbial community scraped off a human tongue, colorized13. Suggest which type of light or electron microscopy should be used to study a given feature of a microorganism a. Dark Fieldi. The spirocheteii. Treponema pallidumiii. Rod shaped bacteria with flagellar
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