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Micro Lab 7
| Question | Answer |
|---|---|
| Pour Plate Method | Serial dilutions of a sample are made and portions of the dilution are "poured" onto a suitable agar growth medium. |
| Streak or Spread Plate | The cells are diluted and then spread onto the medium. Regardless of the plate method, cells are incubated under conditions that permit colonies to develop. |
| It is presumed that each colony develops from a... | single cell |
| What is the reasonable number of cells to be obtained for counting? | 30-300 |
| Water that is not visibly turbid will carry less than 10^7 bacteria per ml, so the highest dilution necessary will be... | 10^6 |
| 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 |
| Turbidity | Turbidity refers to how cloudy a liquid culture looks. The more bacterial cells there are suspended in the broth, the more light they scatter, making the culture appear more turbid. |
| 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. |
| Draw out the dilution scheme | -- |
| 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 |
| Where do we get the numbers for the sample and buffer amounts? | You have to make sure they add up to the ratios. 0.1 mL + 9.9 mL buffer = 1:100 -> 10^2. 1 mL + 9 mL = 1:10 -> 10^1. |
| What if you had a 10^3 ratio? | 0.01 mL + 9.99 mL. (Get checked) |
| Can you go back in a dilution? | No! Choose your factors carefully because you cannot jump from 10^1 back to 10^2. |
| What do microorganisms need to survive? | Suitable water, sunlight, stable temperature, stable pH, aerobic or anaerobic conditions, and a carbon source to build structure and provide energy, They tend to use both macro (C, N, P, etc). and micro-molecules (Fe, Zn, Cu). |
| 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. |
| What is the principle of selection in microbiology? | Selection is the use of selective media to favor (or select for) the growth of one group of organisms over other organisms that might be present. This allows isolation and study of specific microbial groups. |
| What makes Mycophil a selective medium? | Mycophil is selective by pH. It has a pH of ~5, which favors fungal growth since fungi prefer slightly acidic conditions while bacteria generally favor neutral pH. |
| What is TSA (tryptic soy agar) and what type of medium is it? | TSA is a general-purpose, non-selective medium with a pH of ~7 that allows growth of many different organisms, both bacteria and fungi. |
| Why is pH the basis for selection in the fungi from air experiment? | Fungi generally grow at a pH slightly on the acidic side of neutral, whereas bacteria generally favor a neutral pH. This pH preference allows selective growth of fungi on acidic media. |
| What is a colony forming unit (CFU)? | A CFU is a single cell, spore, or even a bit of fungal hyphae that gives rise to a visible colony. It's the unit used to count viable microorganisms, assuming each isolated colony arose from one CFU. |
| What are hyphae (singular: hypha)? | Hyphae are threadlike filaments that form the fungal mycelium. Colonies may develop from a single hypha, which is why we use the term CFU rather than assuming all colonies come from single cells. |
| Why is agar preferred over gelatin as a gelling agent for microbiological media? | Most microorganisms can't utilize agar as a carbon source (so the medium stays solid), and agar has better physical properties for lab work. Gelatin can be degraded by many organisms and is an animal product. |
| What would happen to solid medium if an organism growing on it could use agar as a food source? | The medium would liquefy or dissolve as the organism breaks down the agar polymer for nutrients, destroying the solid support needed for colony isolation. |
| In the fungi from air experiment, what types of microorganisms will grow under the conditions used? | Either bacteria or fungi. The Mycophil selects for fungi (acidic pH), while TSA allows both types to grow for comparison. |
| What is the Citrate test and how does it differentiate bacteria? | The citrate test determines if a bacterium can use citrate as its sole carbon source. Positive organisms turn the medium from green to blue due to alkaline pH from citrate utilization. This is a differential test. |
| What is the MR-VP test and what does it differentiate? | MR-VP consists of two tests: Methyl Red (MR) detects mixed acid fermentation (red = pos), and Voges-Proskauer (VP) detects acetoin production from glucose fermentation (red = pos). Both are differential tests that distinguish fermentation pathways. |
| What is SIM medium and what three things does it test? | SIM tests for: Sulfur reduction (black precipitate = H₂S production), Indole production (red ring with Kovac's reagent), and Motility (cloudiness/growth away from stab line). It's a differential medium testing three different characteristics. |
| What is the Nitrate Reduction test and what does it detect? | Determines if bacteria can reduce nitrate (NO₃⁻) to nitrite (NO₂⁻) or nitrogen gas. Red color after adding reagents = nitrite present (positive). No color change may need zinc addition to confirm complete reduction to gas. It's a differential test. |
| What is EMB agar and is it selective, differential, or both? | EMB (Eosin Methylene Blue) agar is both selective and differential. It selects for Gram-negative bacteria (inhibits Gram-positive) and differentiates based on lactose fermentation. Strong fermenters (like E. coli) show a green metallic sheen. |
| What is Bile Esculin agar and what does it test? | Bile Esculin is both selective and differential. The bile salts select for organisms that can grow in 4% bile. Esculin hydrolysis turns the medium dark brown/black, differentiating esculin-positive organisms. |
| What is Mannitol Salt Agar (MSA) and is it selective, differential, or both? | MSA is both selective and differential. The 7.5% salt selects for halotolerant organisms (mainly Staphylococcus species). It differentiates mannitol fermenters (yellow colonies/medium) from non-fermenters (red/pink), helping identify S. aureus. |
| What is Salt Broth and what does it test? | Salt Broth (typically 6.5% NaCl) is a selective medium that tests an organism's ability to tolerate high salt concentrations. Growth indicates halotolerance. It's used to differentiate enterococci (positive) from other streptococci (negative). |
| What is the Novobiocin test and what does it differentiate? | The novobiocin susceptibility test is differential. It uses a disk containing the antibiotic novobiocin to differentiate staphylococci. |
| What is transformation? | The introduction of foreign DNA into a host cell |
| What are the key characteristics of E. coli strain DH5α? | It lacks the ability to ferment lactose (lac⁻) and is sensitive to the antibiotic ampicillin (amp^s). |
| What is the parent plasmid used in this experiment? | pUC119 is the parent plasmid. |
| How was pRU4x92 created? | By inserting foreign DNA into the multiple cloning site of pUC119. |
| What gene do both pUC119 and pRU4x92 contain? | Both contain ampicillin resistance genes (Amp^r). |
| What is the key difference between pUC119 and pRU4x92? | Only pUC119 has an intact lacZ gene that confers the ability to ferment lactose and produce acid. |
| What is the lacZ gene and what does it do? | The lacZ gene codes for part of the enzyme beta-galactosidase, which allows bacteria to ferment lactose and produce acid. |
| What is the purpose of treating cells with CaCl₂? | To make the cells "competent" - capable of taking up DNA by making the cell membrane more permeable. |
| Why are the cells placed on ice during the competency treatment? | The ice-cold temperature helps make the cells more permeable to DNA uptake. |
| What is the purpose of the heat shock at 42°C for 90 seconds? | The heat shock enhances the cells' uptake of plasmid DNA by making them more permeable. |
| Why is there a recovery period after heat shock? | allow cells to recover from transformation and begin expressing the genes from the plasmid (like ampicillin resistance). |
| What is the purpose of the ampicillin resistance gene in this experiment? | For SELECTION - only cells that took up the plasmid will survive on ampicillin-containing plates. |
| What is the purpose of the lacZ gene in this experiment? | For DIFFERENTIATION - to distinguish between cells transformed with pUC119 (lac⁺) versus pRU4x92 (lac⁻). |
| Why do you plate cells on MacConkey agar with and without ampicillin? | Without ampicillin serves as a control to ensure cells are viable. With ampicillin selects for transformed cells only. |
| What is MacConkey agar and what does it contain? | A differential medium that contains lactose and a pH indicator (neutral red). |
| What color are colonies that ferment lactose on MacConkey agar? | Maroon or pink (due to acid production lowering the pH below 6.8). |
| What color are colonies that cannot ferment lactose on MacConkey agar? | Colorless or amber (pH stays above 8.0). |
| What causes the pH indicator to change color on MacConkey agar? | When lactose is fermented, acid is produced which lowers the pH. Neutral red is maroon below pH 6.8 and amber above pH 8.0. |
| What do maroon colonies on MacConkey agar with ampicillin indicate? | Cells were successfully transformed with pUC119 (have ampicillin resistance and can ferment lactose). |
| What do amber/colorless colonies on MacConkey agar with ampicillin indicate? | Cells were successfully transformed with pRU4x92 (have ampicillin resistance but cannot ferment lactose). |
| What is the goal of plasmid isolation? | To extract and purify plasmid DNA from bacterial cells, separating it from chromosomal DNA, proteins, and cell debris. |
| What property of plasmids makes them useful as cloning vectors? | They can replicate independently of the chromosomal DNA and can carry foreign DNA inserts. |
| What happens when foreign DNA is inserted into the lacZ gene? | The ability to ferment lactose is lost (insertional inactivation). |
| What is a competent cell? | A bacterial cell that is capable of taking up plasmid DNA from solution. |
| Isolation Lysis | Cell lysis - Break open the bacteria using: SDS (detergent) - dissolves cell membranes NaOH (base) - denatures proteins and DNA |
| Isolation Neutralization | Neutralization - Add potassium acetate buffer: Chromosomal DNA and proteins clump together and precipitate Small plasmid DNA stays dissolved in solution |
| Isolation Finishing Steps | Separation - Centrifuge to pellet the precipitated material; keep the liquid containing plasmid Precipitation - Add ethanol to make the plasmid DNA precipitate out of solution Washing & drying - Wash with 70% ethanol and dry to get pure plasmid DNA |
Created by:
smurtab