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Microbio Lab 3
| Question | Answer |
|---|---|
| Original Bacterial Equation | #CFU on plate/uLs plated * 1000 uLs/1 mL * Dilution used to plate = CFU / mL. |
| Suppose you plated 100 µL from a 10⁻³ dilution and counted 50 colonies... | 50/100 * 100 * 10^3 = 500,000 CFU/mL in the original |
| Why should you be counting microbial cells? | Counting microbes helps protect human health and product quality by making sure microbial levels stay safe in food, water, and recreational environments. |
| OD measurement | OD = 0.75 → This is the optical density (turbidity) reading from a spectrophotometer. So, OD = 0.75 → 0.75 × (3 × 10⁸) = 2.25 × 10⁸ cells/mL That’s your original concentration of the undiluted culture. |
| Vol sample | How much of the original (or previous dilution) you added. |
| Vol buffer: | How much sterile diluent (e.g., water or saline) you added. |
| ODF | ODF = product of all dilution steps so far. |
| [Cell/mL] | This is the concentration of cells in each dilution tube. It comes from [cell/mL] = ODF / original concentration |
| You need 400 mL of nutrient broth at 8 g/L. How many grams of powder do you weigh out? | 8g/L×0.400L=3.2g |
| How many grams of agar are needed to make 750 mL of 1.2% agar solution? | 0.012×750mL=9.0g |
| You add 2 mL of a 10 M NaCl stock into 98 mL of water. What is the final concentration? | (10)(2/100)=0.2M |
| If you want 250 mL of 0.5 M glucose from a 2 M stock, how much stock and water do you use? | ((0.5)(250)) / (2) = 62.5 mL stock. Add 187.5 mL water. |
| You plate 0.1 mL of a 10⁻⁴ dilution and count 85 colonies. What is the original concentration? | ((85) / (0.1)) * 10^4 = 8.5 * 10^6 CFU/mL |
| A plate with a 10⁻³ dilution has 310 colonies. Is this usable? | No — countable plates are 30–300 colonies. This one is too high. |
| Why are plate counts expressed as CFU/mL instead of cells/mL? | Because a colony may arise from more than one cell, and we can only count colonies, not individual cells. |
| You spread 0.1 mL of a 10⁻⁵ dilution and get 42 colonies. What’s the concentration? | ((42)/(0.1)) * 10^5 = 4.2×10^7 CFU/mL |
| If all plates are TNTC, what could you do differently? | Make higher dilutions or plate less volume. |
| Why do we start focusing at 4× before going to higher magnification? | Because it gives the widest field of view and prevents damaging the slide/lens. |
| What is the purpose of immersion oil at 100×? | To reduce light refraction and increase resolution. |
| Define: magnification vs resolution. | Magnification = enlarging the image; resolution = ability to distinguish two objects as separate. |
| What is the function of the condenser on a light microscope? | It focuses light onto the specimen. |
| What happens if you use coarse focus at 100×? | You risk crashing the objective into the slide. |
| Place in order: (a) switch to 40×, (b) place slide, (c) use coarse focus, (d) fine focus, (e) immersion oil, (f) 100×. | b → c → a → d → e → f. |
| You are preparing 300 mL of medium with 0.8% NaCl. How many grams of NaCl should you add? | 0.008×300=2.4g |
| How many mL of water should you add to 20 mL of 2 M solution to make a 0.5 M solution? | (2*20) / 0.5 = 80 mL |
| Brightfield Microscopy | The most common and simplest form. Light passes directly through the specimen. If you want to see general morphology and you don’t mind killing/staining cells → Brightfield. |
| Phase-Contrast Microscopy | Enhances contrast in transparent, unstained, living cells. If you want to study living cells, motility, and internal structures without stains → Phase Contrast is more important. |
| General Microscopy Steps | Begin at 10×, focus the specimen with the coarse knob, then switch to 40× and refine focus using only the fine knob. Add a drop of immersion oil before rotating to the 100× objective, then use fine focus and increased light to view under oil immersion. |
| Coarse Focus Knob | Moves the stage up and down quickly for rough focusing, used at low magnification. |
| Fine Focus Knob | Makes small adjustments to sharpen the image, especially at 40× or 100×. |
| Condenser | Focuses light onto the specimen to increase contrast and resolution. |
| Objective Lenses | Typically 4×, 10×, 40×, 100× (oil immersion). Responsible for the primary magnification of the specimen. |
| Immersion oil | used with the 100× oil immersion objective to improve resolution. Placing a drop of oil (which has a similar refractive index to glass) reduces refraction, allowing more light to enter the objective lens and produce a clearer, sharper image. |
| Wavelength Relationship | The wavelength of light used influences resolution, so the shorter the wavelength, the higher the resolution. Electron microscopes have much shorter wavelengths than visible light, so they're much more powerful. |
| Electron | Visualization of internal components (transmission) and three dimensional morphology |
| Who invented the microscope? | Robert Hooke |
| Light vs. Phase | Phase microscopy tends to be more darker and more detailed |
Created by:
smurtab