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Micro Lab Practical
| Question | Answer |
|---|---|
| What are the most likely things to be contaminated in a lab setting? | Cell phones and coats |
| The institution responsible for providing comprehensive health and safety is... | REHS (Rutgers Environmental Health and Safety). |
| Biohazard | An agent of biological origin that can cause disease in humans. Ex: microorganism, toxin, allergen |
| Biosafety | The combined use of laboratory practices, laboratory facilities, and safety equipment to work with potentially infectious organisms. |
| Biosafety Levels | Describes the laboratory facilities required for different infectious agents. |
| Risk Group | Describes the type of organism used in laboratories of varying biosafety levels |
| The BSL we will be using in the microbiology lab is... | BSL-1, which requires the least containment and is the least likely to cause diseases. |
| Four safe work practices associated with the BSL-1 level are to... | always wear your safety goggles, always wear your lab coat, put electronic devices away, and avoid food and drink materials. |
| Any non-glass waste goes into... | The white bucket |
| Place used slides and coverslips in... | Sharps container, red in color and marked with the biohazard symbol |
| Decontamination Methods (Heat) | steam heat, dry heat, incineration |
| Decontamination Methods (Chemical) | bleach, ethanol, hydrogen peroxide, radiation |
| Purpose | What is being done during this exercise? What is the goal? |
| Methods | Citations, Any additional information (e.g. cultures used, incubation times), Written in 3rd person, past-tense |
| Results | Information that you can SEE or OBSERVE form your experiment (e.g. Growth was visible in all four quadrants on the TSA plate) |
| Discussion/Conclusion | Interpretation of your results (The organism was stained red after the Gram stain, thus it is Gram negative). |
| Cardinal Aseptic Rule | Don't open anything until you absolutely need it. Always work next to a Bunsen Burner. |
| Why should you work next to a Bunsen Burner? | to create a sterile work area by using the upward convection current from the flame to push airborne contaminants away from your workspace. |
| Sterile | there are no microorganisms present in the area. |
| Incubate | let the microorganism sit in the environment where it grows best. |
| Culture Media | the nutrients that the microorganism needs to grow. Ex: glucose because it can provide a carbon source. |
| Inoculate | the process of introducing a microorganism or a suspension of microorganisms into a sterile growth medium, such as a petri dish or broth |
| Autoclaving (media) | Uses high-pressure saturated steam (usually 121°C for 15–20 minutes). Kills all microorganisms, including spores. |
| Irradiation (pipettes, plasticware) | Uses gamma rays or UV radiation to sterilize items. Great for heat-sensitive materials, like disposable pipettes or petri dishes. |
| Flame sterilization (loops, needles) | Metal instruments are passed through a Bunsen burner flame. Rapidly kills microorganisms by direct heat. Common in microbiology for loops, inoculating needles, and the tips of glass pipettes. |
| Ethanol sterilization (forceps, surfaces) | Typically 70% ethanol is used. Destroys cell membranes and denatures proteins. Quick, convenient, and good for tools that can’t be autoclaved easily. |
| Only physical method of killing bacteria is... | exposure to UV light. |
| 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). |
| What does it mean when we say that the chemical added should be 1% w/v? | W is the unit for weight (g) and v is the unit for volume (mL). Therefore, 1% w/v means that 1 gram of solute will be dissolved in 100 mL of solvent. The math is done here: 1/100 = 0.01 * 100 = 1%. |
| What should be added to an appropriate all-purpose medium? | 1) dextrose - carbon/energy source, broken down 2) glycolysis casein - keep pH stable, break into amino acids that help with synthesizing proteins 3) NaCl - osmotic balance. otherwise, the microorganism might be at risk of undergoing crenation or lysis. |
| General/All-Purpose Media | Support growth of a wide variety of microorganisms Contain basic nutrients most microbes need Examples: Nutrient agar, Tryptic Soy Broth (TSB) |
| Special Purpose Media | Designed for specific organisms or purposes May be enriched, selective, or differential |
| Liquid Media (Broth): | Nutrients dissolved in water Allow rapid, uniform growth throughout Easy sampling and cell counting |
| Solid Media | Deep: provides little surface for growth, must be stabbed for inoculation, used for testing oxygen limitation Slant: maintenance and storage of pure cultures Plate: used for isolation of colonies, large display of growth but dries quickly |
| Medium | Sterile base that has all the nutrients required for a culture of cells to grow |
| Why is agar used? | It remains solid up to 90 celsius, does not solidify until it is cooled to about 42 celsius to be poured into petri plates, and for most microorganisms, is a nutrient they cannot use. |
| Aseptic technique | set of practices used in the laboratory to prevent contamination of sterile materials, cultures, or yourself while working with microorganisms. Examples: Flaming an inoculating loop before touching a culture. |
| Why didn't we use aseptic technique with the microscopes? | Because we weren't inoculating anything |
| Aseptic Transfer | Innoculation of bacteria is done using a sterile loop for liquid cultures and a needle for solid cultures. The loop or needle is first sterilized using the Bunsen Burner. |
| Dehydrated Trypticase Soy Broth (TSB) | a powdered, nutrient-rich medium that, when rehydrated, supports the growth of a wide range of bacteria and fungi. It is widely used in microbiology for culturing, maintaining, and testing microorganisms in research and clinical labs. |
| Necessary Math for "Preparation of a Culture Medium" Procedure | Using: 30g/L of TSB (30g/1000mL), cross multiply, 3 g to make 100 mL. Agar: 1.5% = 1.5 g for 100 mL. |
| Purpose of "Preparation of Culture Medium" | The purpose of this experiment was to make TSA slants. |
| Purpose of "Slant to Slant Transfer" | To safely transfer the contents of one slant to another. |
| Slant to Slant Transfer Predictions | I predicted that my subculture would look like my parent culture, Sarcina aurantiaca, orangish with a fuzzy texture. |
| Common incubation temperatures | 37 degrees celsius (body) and 25 (room). They tend to be incubated from around 24 to 48 hours. |
| QSP | A streaking method used to isolate pure colonies of microorganisms from a mixed culture. The agar plate is divided into four quadrants, and an inoculating loop is streaked across them in a systematic way to dilute the microbial sample. |
| Do not's (QSP) | Don't overload the loop. will thick smear of growth → no isolated colonies. Don't forget to flame between quadrants → everything overgrows. Do not streak back into the first quadrant too many times → brings too many cells forward. USE THE THIN SIDE. |
| Pure culture | A culture containing only the progeny of a single microbial cell, consisting of one species and strain. |
| Mixed culture | A culture that contains several different microbial species and strains. |
| Isolated colony | A single visible colony that grows from one isolated cell on an agar plate, separated from other colonies; assumed to have developed from a single cell but not yet confirmed as a pure culture until subcultured in the absence of other cell types. |
| Purpose of "Quadrant Slant Plating" | To isolate bacterial colonies |
| In QSP, each parent cell reproduces to form... | A colony. |
| During QSP, the loop only goes into the test tube... | ONCE |
| Colonies | Visible masses of growth that represent division of one parent cell or colony forming unit (CFU). To evaluate colony growth, observe characteristics such as pigment, margin, and elevation of colonies. |
| How do you know a culture is contaminated? | You know a culture is contaminated by observing visual changes like cloudiness or turbidity, visible growth like clumps or filaments, and color changes in the medium. The presence of different colony types indicates contamination. |
| Pour Plate Method | Serial dilutions of a sample are made and portions of the dilution are "poured" onto a suitable agar growth medium. To quantify the number of bacteria in a liquid sample, allowing for growth both on and within the agar. |
| 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. Streak isolates colonies while spread does enumeration. |
| It is presumed that each colony develops from a... | single cell |
| What is the reasonable number of cells to be obtained for serial dilution 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 |
| Vortexing | Vortexing a buffer solution is a standard laboratory procedure used to quickly and uniformly mix solutes |
| What sample did we use for serial dilution? | An Ecoli Sample |
| 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 * 1000 * 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] = original concentration / ODF |
| 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. |
| Can you go back in a dilution? | No! Choose your factors carefully because you cannot jump from 10^1 back to 10^2. |
| Purpose of serial dilution experiment | the purpose was to dilute a sample to the point where the number of bacterial colonies could be visibly counted. |
| 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? | 1.2% means 1.2 g per 100 mL of solution. So 1.2/100 = x/750, cross multiply 1.2*750/100 = 9.0 g. |
| You add 2 mL of a 10 M NaCl stock into 98 mL of water. What is the final concentration? | Use M1,V1 = M2,V2 equation (2)(10) = (x)(98+2) (2*10)/100 = 0.2 M |
| 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. |
| 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 |
| What are the best targets for antimicrobial agents? | When considering cellular targets for antimicrobial agents, the best targets are those that are essential to the microbe’s survival or replication but are absent or significantly different in human cells. Ex: cell wall synthesis, cell membrane, etc. |
| Physical Methods of Microbial Control: | Moist heat, refrigeration & freezing, filtration, osmotic pressure, UV light |
| Chemical Methods of Microbial Control: | Phenol and phenolics, alcohols, halogens, enzymes, heavy metals, antibiotics |
| Which concentration of alcohols is best for microbial control? | The most effective concentration of alcohols for microbial control is usually 70% ethanol. 70% works better than 100% because water is essential for denaturing proteins and it has slower evaporation, stays on the surface longer. |
| Examples of Halogens used for microbial control | Iodine tablets, iodophores, chlorine treatment, bleach. |
| Examples of Enzymes being used for microbial control | Lysozyme is used to reduce the number of bacteria in cheese. |
| Examples of heavy metals used for microbial control | Ag, Hg, and Cu |
| Antiseptics | Work only on cells that are actively metabolizing and dividing, Are bacteriostatic – stopping cell division as long as the bacteria are in direct contact with the antiseptic, Safe for use on tissue |
| Disinfectants | Effective against spores, must be bacteriocidal (killing bacteria), designed for use on formites, take time after usage to work properly, general time is 10-15 minutes |
| Most Resistant to Least Resistant to Microbial Control | prions, endospores of bacteria, mycobacteria, gram negative bacteria, gram positive bacteria, viruses with lipid envelopes |
| What did you have to include in your drawing of microscopic specimens? | Name of organism, magnification, type of microscopy used, a size or scale marker, a few descriptive words as appropriate. |
| Antimicrobial Agents Types: | antibacterial agents can be classified as bacteriostatic, bacteriocidal, and bacteriolytic by observing their effects on cultures. |
| Bacteriostatic Agents | inhibit important biochemical processes and bind weakly |
| Bactericidal Agents | bind tightly and kill the cell without lysis |
| Bacteriolytic Agents | kill by lysis (for example, detergent). |
| Match the Chemical Antimicrobial Agents | Sterilants → bactericidal, bacteriolytic Disinfectants → best is10% bleach, bactericidal, bacteriolytic, used non-living surfaces Sanitzers → bacteriostatic Antiseptics (gemicides) → bacteriostatic, can be used on living tissue, 70% ethanol. |
| Sterile Vs. Aseptic Techniques | Sterile means completely free of all microorganisms, while aseptic refers to techniques that prevent contamination |
| Pasteurization Vs. Autoclaving | Pasteurization uses moderate heat to reduce pathogens (doesn't kill all microbes), while autoclaving uses high-pressure steam to achieve sterility |
| Heat, UV Light, Gamma radiation, and Filtration: | Different physical methods to kill or remove microorganisms. Additional tips include → moist heat is the best, some bacteria can repair themselves when hit with UV light, a funnel would have to be smaller or equal to 0.4 micrometers. |
| Sterilization | Completely free of all living microorganisms, including bacteria, viruses, fungi, and spores. |
| Aseptic | Techniques and conditions that prevent contamination by microorganisms; maintaining a sterile environment during procedures. |
| Pasteurization | Heat treatment that reduces the number of pathogenic microorganisms to safe levels without achieving complete sterility (typically 63°C for 30 minutes or 72°C for 15 seconds). |
| Autoclaving | Sterilization method using pressurized steam at high temperature (typically 121°C at 15 psi for 15-20 minutes) to kill all microorganisms including spores. |
| Autoclave Function Test | uses Bacillus spores. Failure of spores to be killed indicates a malfunction. |
| Home Equipment | pressure cookers can achieve sterilizing temperatures due to pressure. Ovens use dry heat, which requires longer exposure times. |
| Boiling Water | does NOT sterilize (does not kill spores). Kills pathogens in water. |
| 100μLs added to 900μLs is a total of 1000 μLs. This is... | 100 in 1000 therefore, a 1:10 dilution (10^1 ) dilution. |
| 10μLs added to 990 μLs is a total of 1000μLs. This is... | 10 in 1000 therefore, a 1:100 (10^2 ) dilution. |
| Total Volume/Dilution Amount = | Volume needed of agent being diluted |
| Total Volume - Volume needed of agent being diluted = | Volume of diluting solution |
| What is the main difference revealed by Gram staining? | Distinguishes bacteria as Gram-positive (purple) or Gram-negative (pink) based on cell wall structure. |
| What does simple staining show? | The shape, size, and arrangement of bacterial cells using a single dye. |
| Acid-Fast Stain | Detects Mycobacterium species that have waxy mycolic acid cell walls. |
| Name and describe three main types of media. | Selective: Supports growth of certain microbes only. Differential: Shows visible differences (e.g., color changes). Enriched: Contains nutrients for fastidious organisms. |
| Ultraviolet (UV) radiation | A form of electromagnetic radiation with lethal effects on microorganisms; has longer wavelength and less penetrating power than gamma radiation. |
| Germicidal activity | The ability to kill microorganisms; for UV light, this activity is greatest at 260 nm wavelength. |
| Deoxyribonucleic acid (DNA) | The genetic material of cells that has maximum absorption of UV light at 260 nm; the primary target of UV radiation damage. |
| Thymine-thymine dimers | DNA damage caused by UV radiation where adjacent thymine bases become crosslinked together, inhibiting DNA replication. |
| Endospores | Resting cells formed by certain bacteria (such as Bacillus and Clostridium) in which DNA is not actively synthesized, making them resistant to UV radiation. |
| Penetrating power | The ability of radiation to pass through materials; UV has limited penetrating power compared to gamma radiation, making it useful primarily for sterilizing air and surfaces. |
| Total magnification formula | the power of the objective lens multiplied by the power of the ocular lens (eyepiece). For example, if you have a 40x objective lens and a 10x eyepiece, the total magnification is 400x (40 x 10 = 400x). |
| What's the total magnification for a 10x lens? | 10 x 10 = 100 |
| What's the total magnification for a 40x lens? | 40 x 10 = 400 |
| What's the total magnification for a 100x (oil immersion) lens? | 10 * 100 = 1000 (oil immersion) |
| 100x Magnification to Scale | 10 um |
| 400x Magnification to Scale | 2.5 um |
| 1000x Magnification to Scale | 1.0 um |
| Purpose of Staining Experiment | The purpose of this experiment was to perform a negative stain and gram stains on different specimens. |
| Specimens Used for the Staining Experiment | Unknown, Bacillus subtilis (negative stain), Micrococcus luteus (gram positive control), Pseudomonas flourescens (gram negative control) |
| Micrococcus luteus | Cocci, purple cells. The purple color led to the identification of Micrococcus luteus as gram positive, meaning it has a thick peptidoglycan wall and retained the crystal-violet better. |
| Pseudomonas flourescens | Bacillus, pink cells. The pink color led to the identifcation of Pseudomonas flourescens as gram negative, meaning it has a thin peptidoglycan wall and was more susceptible to the counterstain. |
| Bacillus subtilis | The negative stain experiment was successful, because it correctly showcased the shape and structure of the cells without any of the additional details found in gram stains. Colorless, rod-shaped |
| Common Cell Types (In Staining) | The gram positive characteristic is unique to some bacteria, yeasts, and a few molds. Most other living cells are gram negative or gram variable. A few other are gram non-reactive, like Mycobacterium. |
| Gram-positive bacteria | the cell wall of Gram-positive bacteria has peptidoglycan, a polysaccharide with peptide cross-linkages. The crystal violet can't pass through peptidoglycan during decolorization, as the solvent causes the thick peptidoglycan to contract and thus retain. |
| Gram-negative bacteria | peptidoglycan is a minor component and there is also a proteolipid, neither of which prevents the stain loss. If the thick peptidoglycan is removed from a gram-positive cell, the cells will not retain the stain complex and will appear Gram-negative. |
| General Protocol for Gram Stain | 1) Primary stain 2) Mordant 3) Decolorizing agent 4) Counterstain |
| Primary Stain | crystal violet, stains all cells purple. |
| Mordant | enhance a formation between dye and the bacterial cell. An iodine solution serves is the mordant. |
| Decolorizing agent | acetone, alcohol, etc. Removes primary stain from Gram-negative and "dehydrates" the Gram-positive. |
| Counterstain | recolors cells that have lost the primary stain after alcohol treatment |
| Why is decolorization a critical step in the procedure? | Decolorization is a critical step in procedures like Gram staining because it differentiates between cellular components, such as bacteria, by selectively removing a primary stain from some while leaving it on others. |
| What did we used as a decolorizer? | Decolorizer: Acetone-Ethanol (50:50 - Red squirt bottle) |
| Example of Gram-Negatives | Enterobacter aerogenes Escherichia coli Pseudomonas fluorescens Citrobacter freundii |
| Example of Gram-Positives | Bacillus subtilis Staphylococcus epidermidis Micrococcus luteus Enterococcus durans |
| Why do we use a wax pencil? | The wax is water-resistant, so it doesn't wash off during staining. |
| Heat Fixing | To heat fix, first let the smear air-dry completely. Then, pass the slide quickly through the flame of a Bunsen burner in a P shape. We heat fix to preserve the bacteria shape so they don’t wash off or distort during staining. |
| Brightfield Microscopy with Gram Stains | Examine the slides under bright-field with no cover slip. Focus the slide beginning at 100x total magnification (10x objective). You will need the oil-immersion objective (1000X) to judge the quality of your Gram stain. |
| Negative Stain | determines cellular morphology and to view capsules. Since the cells themselves are not stained, their morphology is not distorted in any way. The nigrosin provides a dark background against which the shapes of the unstained cells are clearly visible. |
| Negative Stain Procedure | Place a drop of nigrosin on a slide, mix in a small amount of organism, and use another slide to spread the mixture into a thin film. Let it air dry completely, then observe under microscope. |
| Gram Stain Vs Negative Stain | Gram stain: Differentiates bacteria into two major groups (Gram-positive and Gram-negative) based on their cell wall structure Negative stain: Determines cellular morphology and allows visualization of capsules without distorting the cells. No colors. |
| After correct Gram staining, the gram-negative cells will appear _____, whereas the gram-positive cells appear _____ | pink, purple |
| Your Gram stain is complete and correct. Which of the following statements would apply to the image you see? Select all that apply | you can observe: -gram-neg bacilli -& gram-pos cocci |
| After you complete the staining, you observe only gram negative cells. Which of the following statements would apply to the image you see? | This image could be explained by omission of the Gram's iodine (mordant) step, causing all cells to appear gram-negative. the primary crystal violet stain won't be fixed to the cell walls and will be washed away during the decolorization step. |
| After you complete the staining, you realize that you did not perform the safranin step. Which of the following statements would correctly describe what you would see? | Gram-negative organisms might not be visible. Gram pos would be purple and Gram neg would be colorless. |
| After you complete the staining, you realize that you performed the decolorizer step for 10 minutes instead of 10 seconds! Which of the following statements would correctly describe what you would see? | Gram-positive organisms might incorrectly stain pink. Both gram pos and gram neg would be stained pink |
| After you complete the staining, you realize that you did not use the alcohol bottle at all. Which cells would appear purple? Select all that apply. | Gram-negative cells Gram-positive cells |
| Why do all my cells appear pink, including the Gram-positive control? | You over-decolorized by leaving the decolorizer on too long or using a smear that was too thin. |
| Why do all my cells appear purple, including the Gram-negative control? | You under-decolorized by not applying the decolorizer long enough or the smear was too thick. |
| Why do my Gram-positive cells appear pink even though I followed all the steps? | You likely skipped or didn't apply the iodine mordant for the full minute. |
| Why does the same organism show both purple and pink cells on my slide? | Your culture is too old (over 24 hours) or you have a contaminated mixed culture. |
| Why are my cells barely visible and very faintly colored? | Your crystal violet or safranin reagents are too old, too dilute, or you didn't stain long enough. |
| Why can't I see any cells on my slide at all? | You didn't heat-fix the slide properly, so the smear washed off during staining. |
| Why are my bacteria clumped together instead of individual cells? | You used too much culture material or didn't mix and spread it properly with water. |
| Why is my slide background stained purple instead of clear? | You didn't wash off excess crystal violet thoroughly enough after the primary stain step. |
| Why are my known control organisms showing the wrong Gram reaction? | Your control cultures are contaminated, too old, or your reagents have expired. |
| How long should I decolorize to avoid both over and under-decolorization? | Decolorize until the purple color just stops draining from the slide into the waste container. |
| What happens if I forget to blot dry after washing off the iodine? | Excess water will dilute the decolorizer, causing under-decolorization and false Gram-positive results. |
| Why do my results vary every time I stain the same organism? | You're inconsistent with timing, especially during the critical decolorization step. |
| What causes Gram-positive bacteria to lose their ability to retain crystal violet? | Old cultures, damage to cell wall |
| Why is it critical to use cultures that are 18-24 hours old? | Older Gram-positive cultures may have damaged cell walls and appear Gram-negative. |
| What's the most common mistake that causes false Gram-negative results? | Over-decolorization, which strips the crystal violet-iodine complex from Gram-positive cells. |
| What's the most common mistake that causes false Gram-positive results? | Under-decolorization, which leaves the crystal violet-iodine complex in Gram-negative cells. |
| What is selective media? | Media that favors (or selects for) the growth of one group of organisms over other organisms that might be present. |
| What is the pH of Mycophil medium and what does it select for? | Mycophil has a pH of ~5, which selects for fungi (which prefer slightly acidic conditions) over bacteria (which prefer neutral pH). |
| What is TSA and what is its purpose? | TSA (Tryptic Soy Agar) is a general-purpose, non-selective medium with a pH of ~7 that allows growth of many different organisms. |
| What is the basis for selection in the air sampling experiment? | pH is the basis for selection. Fungi grow at pH slightly on the acidic side of neutral, whereas bacteria generally favor a neutral pH. |
| What does CFU stand for and what does it represent? | CFU stands for Colony Forming Unit. It represents a single cell, spore, or bit of fungal hyphae that can grow into a visible colony. |
| What are hyphae? | Hyphae (singular: hypha) are threadlike filaments that form the fungal mycelium. |
| What is agar and what is its primary function? | Agar is a gelling agent derived from seaweed (a polysaccharide with galactose as the basic building block) that provides solid support for bacterial growth. |
| Why can't most microorganisms use agar as a carbon source, and what would happen if they could? | Most microorganisms can't utilize the polysaccharide in agar as a carbon source. If they could use it as food, the solid medium would liquefy as the organism consumed the agar. |
| At what temperature does agar powder go into solution, and what temperature does it solidify? | Agar goes into solution when heated to boiling (100°C) and solidifies when cooled to ~42°C. |
| What happens to agar once it solidifies at 42°C? | Once agar solidifies at 42°C, it remains solid until it is heated back to 100°C. This allows incubation at elevated temperatures without the medium liquefying. |
| Why is agar preferred over gelatin for microbiological media? | Agar has two key advantages: (1) most microorganisms can't utilize it as a carbon source, so the medium stays solid, and (2) it remains solid at incubation temperatures, whereas gelatin would liquefy. |
| Flood | add substance til circle is full, circles should not overlap, wait, hold perpendicular to you add water til runoff is clear |
| Blot | Blot dry, make a sandwich |
| Nigrosin Purpose (gram negative) | Nigrosin stains the background, leaving Gram-negative bacteria visible and well-defined. |
| Three Common mistakes in A Microscopy Drawing | they weren’t drawn to scale, description should be full sentences, drew the wrong organism. |
| Three reasons why we use a control | make sure the procedure was performed correctly, to compare against the unknown, determine whether or not the reagents worked. |
| stereomicroscope | low-magnification microscope that provides a three-dimensional view of the surface of larger, opaque specimens. |
| What is the general workflow for identifying an unknown microorganism? | Use mixed culture, isolate the unknown using quadrant streak plate and slant transfer, perform wetmount observation, conduct Gram staining, run biochemical tests (enzymatic and metabolic), and finally compare results to data tables for identification. |
| What determines a microorganism's metabolic capabilities? | genetic composition determines its metabolic capabilities. The complete set of enzymes a microorganism possesses is encoded by its DNA, which determines what metabolic reactions it can perform. Gene expression also depends on available substrates. |
| What are the three main methods for visualizing metabolic reactions? | 1) Inherently visible reaction products (e.g., bubbles in catalase test), 2) Inclusion of pH indicators to observe color changes from acidic/alkaline byproducts, 3) Addition of chemical compounds after growth to modify products into colored compounds. |
| What is an example of an inherently visible reaction product test? | The catalase test, where the reaction produces bubbles (oxygen gas) that can be directly observed without adding any indicators or chemicals. |
| How do pH indicator methods work in metabolic testing? | pH indicators change color based on acidic or alkaline byproducts produced during metabolism. Examples include Phenol Red in lactose/glucose broths, citrate slant, and urease test. |
| What is the purpose of adding chemical compounds after organism growth? | When direct pH indicator observation isn't possible, chemical compounds are added to react with metabolic products or intermediates to form colored compounds. Example: Kovac's reagent or oxidase reagent to detect indole presence. |
| What factors affect the rate of enzyme-catalyzed reactions? | Temperature, enzyme concentration, substrate concentration, and the enzyme's affinity for the substrate all influence the reaction rate. |
| How do enzymatic capabilities relate to an organism's phenotype? | A microorganism's physiological properties are largely determined by its enzymatic capacities (phenotype reflects genotype). These capabilities also determine its habitat and ecological relationships with other organisms. |
| What does the catalase test check for, and what are some possible outcomes? | It checks for the enzyme catalase, which breaks down hydrogen peroxide (H₂O₂) into water and oxygen. Positive result: bubbles form (e.g., Staphylococcus). Negative result: no bubbles (e.g., Streptococcus). |
| What does the oxidase test check for, and what are some possible outcomes? | It checks for the enzyme cytochrome c oxidase, part of the electron transport chain. Positive result: color change to dark purple/blue within seconds (e.g., Pseudomonas). Negative result: no color change (e.g., Escherichia coli). |
| 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). To differentiate, you add zinc: Red after zinc → Nitrate still present → Negative (organism did NOT reduce n |
| What does the Phenol Red Glucose test check for, and what are some possible outcomes? | Differential. Tests if a bacterium can ferment glucose and produce acid or gas. Acid positive: yellow (pH less than 6.6, e.g., E. coli) Alkaline/negative: red medium (no fermentation, e.g., Pseudomonas) Gas production: bubbles in the Durham tube |
| What does the Phenol Red Lactose test check for, and is it differential or selective? | It is differential and tests whether a bacterium can ferment lactose. Acid positive: yellow medium (pH less than 6.6, e.g., E. coli) Alkaline/negative: red medium (no fermentation, e.g., Pseudomonas) Gas production: bubbles in the Durham tube |
| What is the purpose of 6.5% NaCl TSB, and is it differential or selective? | It is selective for bacteria that can tolerate high salt concentrations, such as Enterococcus species. Positive result: growth/turbidity. Negative result: no growth. |
| What does the Bile Esculin test check for, and is it differential or selective? | Selective: bile inhibits most Gram-positive bacteria except enterococci and some streptococci Differential: tests ability to hydrolyze esculin. Pos: dark brown/black (e.g., Enterococcus faecalis). Negative result: light or unchanged. |
| What is EMB agar used for, and is it differential or selective? | Selective: inhibits growth of most pos bacteria, favoring neg. Differential: differentiates bacteria based on lactose fermentation: Strong fermenters: metallic green sheen (e.g., E. coli) Weak: pink (e.g., Enterobacter) Non: colorless |
| What does the Novobiocin test check for, and is it differential or selective? | It is differential and tests whether bacteria are susceptible or resistant to novobiocin, an antibiotic. Differentiates between staplycoccus species. Neg: very small zone Pos: pronounced zone |
| What is Mannitol Salt Agar used for, and is it differential or selective? | Selective: high salt (7.5% NaCl) inhibits most bacteria except staphylococci. Halotolerant organisms. Differential: tests mannitol fermentation: Pos: yellow colonies Negative: pink/red colonies/medium |
| Fluid Thioglycollate Medium | a liquid medium that creates an oxygen gradient to determine a bacterium’s oxygen requirements. Has outcomes like obligate anaerobe, etc. |
| Obligate aerobe | Bacteria that require oxygen for growth and survive only at the top of a medium. |
| Obligate anaerobe | Bacteria that cannot tolerate oxygen and grow only at the bottom of a medium. |
| Facultative anaerobe | Bacteria that can grow with or without oxygen, but grow better in its presence. |
| Microaerophile | Bacteria that require low oxygen levels and grow just below the surface of a medium. |
| Aerotolerant | Bacteria that do not use oxygen but can tolerate its presence, growing evenly throughout a medium. |
| Draw all of the outcomes of the fluid thioglycollate test | -- |
| 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. |
| What are the main similarities between pUC119 and pRU4X92 plasmid maps? | Both plasmids contain an ampicillin resistance gene (Amp r) for selection and have multiple cloning sites with several shared restriction enzyme cut sites including EcoRI. |
| What are the main differences between pUC119 and pRU4X92 plasmid maps? | pUC119 is 3.2 Kb in size with a lac Z' region and M13 origin. pRU4X92 is larger with an M13 IG region and a different origin (Ori). The restriction sites are arranged differently in each plasmid's multiple cloning site. |
| What is the purpose of ethidium bromide (EtBr) in gel electrophoresis? | It intercalates into DNA and fluoresces under UV light, allowing visualization of DNA bands |
| What percentage agarose gel is appropriate for the DNA fragments in this restriction digest? | 1% agarose gel |
| What is the purpose of gel loading buffer (GLB)? | Contains tracking dye and increases sample density so it sinks into the wells |
| What temperature and duration are used for the restriction enzyme digestion? | 37°C for 40-45 minutes |
| What factors determine the rate of DNA migration through an agarose gel? | Size and shape of the molecule; smaller molecules and compact molecules move faster than large, loose ones |
| What is the recognition sequence for HindIII? | 5' AAGCTT 3' / 3' TTCGAA 5' (produced by Haemophilus influenzae) |
| What is the recognition sequence for EcoRI? | 5' GAATTC 3' / 3' CTTAAG 5' (produced by Escherichia coli) |
| What happens to circular plasmid DNA when cut by ONE restriction enzyme? | It becomes linearized (one linear piece) |
| What happens to circular plasmid DNA when cut by TWO restriction enzymes? | It produces two linear fragments |
| Why does DNA migrate toward the anode (positive terminal) in gel electrophoresis? | Because DNA is negatively charged (it's an anion) and moves toward the positive terminal |
| How do closed circular plasmids compare to open circular (nicked) plasmids in migration speed? | Closed circular (supercoiled) plasmids move FASTER than open circular plasmids of the same size |
| How much sample is loaded into each well? | Approximately 15 μl of sample (after mixing with 2 μl GLB from the 20 μl total) |
| Where are the EcoRI and HindIII restriction sites located on both plasmids? | In the multiple cloning site (MCS) of each plasmid |
| How can you identify which plasmid you isolated based on the gel results? | By comparing the number and sizes of fragments from each digest to the predicted pattern; each plasmid will have different fragment sizes when cut with the same enzymes |
| Why must gel boxes be handled with gloves? | Ethidium bromide is a mild carcinogen that can contaminate gel boxes. Gloves protect against exposure to this hazardous chemical. |
| Why is the lambda DNA size marker (ladder) essential in gel electrophoresis? | Lambda DNA markers provide fragments of known sizes that serve as references for estimating the size of unknown DNA fragments. Without them, you cannot determine fragment lengths or accurately identify your plasmid. |
| What could cause no bands to appear for your plasmid samples? | Possible causes include: insufficient DNA loaded, DNA degraded during isolation, incomplete digestion, forgetting to add gel loading buffer, samples not loaded into wells properly, or excessive electrophoresis time causing DNA to run off the gel. |
| Why might uncut plasmid show multiple bands? | Plasmids can exist in different forms: supercoiled (fastest migration), open circular/relaxed (slowest), and linear (intermediate). These different conformations migrate at different rates even though they're the same molecule. |
| Explain the prediction table | pUC119: All digests produce 1 band at 3.2 kb (both restriction sites at same location). pRU4X92: Single digests produce 1 band (linearized plasmid), double digest produces 2 bands (3.2 kb + insert size) because insert separates the two restriction sites. |
| Do the Math for the Water needed in Gel Electrophoresis. To prepare 350 mL of 1x TAC solution from a 10x stock: | Water Needed: TAE Stock (C1) = 10x TAC Solution (C2) = 1x Volume 2 (V2) = 350 mL C1V1 = C2V2 10x * V1 = 1 * 350 mL (1x * 350 mL) / (10x) = V1 V1 = 35.0 mL Water Result = 350 mL - 35.0 = 315 mL |
| Do the Math for the Agarose needed in Gel Electrophoresis | Goal for agarose = 1% of 40 mL 1 g / 100 mL = x / 40mL, then cross-multiply. x = 0.4 grams of agarose needed. |
| Tryptone | Provides peptides and amino acids as nitrogen and carbon sources for bacterial growth. |
| Yeast extract | Supplies vitamins, minerals, and additional nitrogen compounds essential for metabolism. |
| Lactose | Serves as a fermentable carbohydrate that can be used as a carbon and energy source by certain bacteria. |
| Mannitol | Acts as an alternative fermentable sugar for carbon and energy, useful for differentiating bacterial species. |
| Dipotassium phosphate | Buffers the medium to maintain pH stability and provides phosphate for nucleic acid synthesis. |
| NaCl | Maintains osmotic balance and provides essential sodium and chloride ions for cellular functions. |
| Tip for Dilution Diagram | Make sure that there are a total of 5 tubes, even if you accomplish the goal by the 4th! |
| Divide Sign on Micropipettes | Tells you what range of nanometers the pipette can dispense. Pipettes are tall at max volume and short at min, |
| First Stop | Helps you draw liquid |
| Second Stop | Helps you eject liquid |
| How to use pipette | Set it accordingly, then push it down to the first stop. While pressing down, insert into the liquid. Then draw back slowly. To transfer, push down to the first stop, then the second. |
Created by:
smurtab