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Microbio Lab Midterm
| Question | Answer |
|---|---|
| What are the most likely things to be contaminated in a lab setting? | Cell phones and contaminated |
| 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. |
| 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? | dextrose - carbon/energy source, broken down, glycolysis casein - keep pH stable, break into amino acids that help with synthesiz proteins 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 soldify 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. |
| Summarize making the agar slants | combine dehydrated TSB and 1.5% agar with 100 ml distilled water, then microwave until the agar dissolves. The cooled medium is dispensed into test tubes (7 ml each) and autoclaved at 121°C for 15 minutes. |
| Necessary Math for Procedure | Using: 30g/L of TSB (30g/1000mL), cross multiply, 3 g to make 100 mL. Agar: 15% = 1.5 g for 100 mL. |
| Purpose of "Preparation of Culture Medium" | The purpose of this experiment was to make TSA slants. |
| Summarize slant to slant transfer | Heat needle, remove cap (without putting it down), remove a small amount of surface growth from slant, streak this inside the fresh agar slant from bottom up, replace cap and heat needle. |
| 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. Dont forget to flame between quadrants -> everything overgrows. Do not streak back into the first quadrant too many times. repeated dragging brings too many cells forward. |
| Summarize QSP Method | Divide plate, Use a sterile loop to place mixed culture in quadrant 1 and streak back and forth heavily, then streak from quadrant 1 into quadrant 2 with fewer strokes, repeat the process, incubate at 34 c. Flame between quadrants. |
| 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 |
| What are the steps of the serial dilution procedure? | Prepare your dilutions by placing buffeted water into test tubes according to the dilution scheme. Vortex each after doing so. Distribute 1 mL of each into Petri dishes, pour liquified agar inside, replace lid and swirl as you wait for them to harden. |
| 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. |
| 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. |
| 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 |
| 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 were the purposes of the two microscope experiments done in our class? | The first was to observe protists from Passion Pond using brightfield microscopy (only 40x), and the second was to observe known and unknown specimens using Phase microscopy (100x). |
| 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 vs. 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 |
| 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) |
| 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. |
| Dark-field Microscopy | Illuminates specimens with oblique light so they appear bright against a dark background, ideal for observing live, unstained microorganisms. |
| Electron Microscopy (TEM & SEM) | Uses electron beams instead of light to visualize cellular ultrastructure (TEM) or detailed surface topography (SEM) |
| Eyepiece (Ocular Lens) | The lens you look through; typically magnifies the image 10×. |
| What is the function of the revolving nosepiece? | Holds and rotates the objective lenses, allowing easy magnification changes. |
| What is the stage on a microscope? | The flat platform where slides are placed for viewing. |
| What do the stage clips or mechanical stage do? | Hold the slide securely and allow smooth movement for precise positioning. |
| What is the function of the arm? | Connects the base and upper parts; used for carrying the microscope safely. |
| What does the base of the microscope do? | Supports the microscope and contains the light source or mirror. |
| What is the purpose of the light source? | Provides the light needed to view the specimen; can be a built-in lamp or external mirror. |
| Diaphragm (Iris Diaphragm) | Controls the amount of light that passes through the specimen. |
| What is the body tube? | Maintains the correct distance between the ocular and objective lenses. |
| What does the head of the microscope contain? | Houses the optical components (ocular and objective lenses). |
| What is the function of the microscope slide? | A rectangular glass plate that holds the specimen for viewing under the microscope. |
| What’s the first step before focusing a brightfield microscope? | Place the slide securely into the slide clip on the stage. |
| Which objective lens should you start with when focusing? | Begin with the 10× objective lens, ensuring the specimen is centered under the lens. |
| How do you initially bring the specimen into view? | While looking at the microscope, use the coarse focus to raise the stage until it stops. |
| How should you adjust the condenser before focusing? | Raise the condenser to its highest position, set the condenser dial to “0”, and close the condenser diaphragm. |
| How do you locate the specimen under the microscope? | While looking through the microscope, lower the stage slowly using the coarse focus until the image appears. |
| What should you do once the specimen is nearly in focus? | Use the fine focus knob to sharpen the image. |
| How do you begin adjusting for maximum resolution? | Close the field diaphragm until the edges come into view. |
| What should you do when you see the diaphragm edges? | Adjust the condenser height to make the edges sharp and centered. |
| Once the diaphragm edges are sharp, what’s next? | Open the field diaphragm until its edges align with the edge of the field of view. |
| What must you do to increase magnification? | Rotate the nosepiece to the 40× objective and adjust the fine focus slightly. |
| What if the image looks cloudy or disappears at 40×? | Clean oil off the 40× objective and refocus. |
| What’s the correct procedure to use the 100× objective? | After focusing at 40×, rotate the nosepiece so no lens is over the slide. Place a drop of immersion oil on the specimen. Rotate the 100× objective into place. |
| How do you sharpen the image under oil immersion? | Use the fine focus knob carefully for the clearest image. |
| Microscopy Steps Condensed | slide, 10x, coarse raise stage, condensor high, lower stage til image, fine focus, close diaphragm for edges, open, fine, 40x, 100x |
| Make sure you can arrange all the parts on the picture of the microscope diagram you took | -- |
| QSP: Why didn’t you get isolated colonies? | The inoculum was too heavy, so too many cells were deposited in the first quadrant. Also, the loop may not have been sterilized properly between quadrants, preventing proper dilution. |
| QSP: Why did growth appear in all quadrants? | The loop may have carried too many cells over due to insufficient sterilization. Streaks that are too close together can also prevent proper dilution. |
| QSP: Why is there no growth on the plate? | The organism may not have been viable or was applied in too small an amount. Alternatively, the agar surface could have been too dry or unsuitable for that microbe. |
| QSP: How do you prevent contamination? | Use proper aseptic technique, such as flaming the loop and disinfecting surfaces. Keep the plate closed as much as possible and avoid touching non-sterile surfaces. |
| QSP: Why are streaks uneven or smudged? | Pressing the loop too hard can gouge the agar, causing smears. Wet agar or inconsistent hand movement can also spread cells unevenly. |
| QSP: Why flame the loop between quadrants? | Flaming sterilizes the loop and reduces the number of cells carried over. This ensures proper dilution and helps achieve isolated colonies. |
| SD: Why did colony counts not decrease as expected? | The previous dilution may have been done incorrectly, so too many cells carried over. Inaccurate pipetting or not mixing the solution well can prevent proper dilution. |
| SD: Why are there no colonies on the plate? | The inoculum may have been too small, or cells were non-viable. Another possibility is that the dilution was too high, leaving too few cells to grow. |
| SD: Why are there too many colonies to count? | The dilution was likely too low, leaving a very dense cell population. Pipetting errors or not mixing the previous dilution can also cause excessive growth. |
| SD: Why are colonies unevenly distributed on the plate? | The spread technique may have been inconsistent or the plate tilted. Uneven mixing of the dilution before plating can also lead to clustering of cells. |
| M: Why is the image blurry? | The objective lens may not be properly focused, or the slide is not correctly positioned. Dirt or oil on the lens can also reduce clarity. |
| M: Why is there low contrast? | The light source may be too dim, or the diaphragm/iris is not adjusted correctly. |
| M: Why can’t I see any cells? | The sample may be too sparse or not properly prepared on the slide. Using the wrong objective lens or insufficient illumination can also make cells invisible. |
| M: Why is there uneven illumination? | The light source may be misaligned or the condenser is not adjusted properly. Dirt on the lens or slide can also cause uneven lighting. |
| M: Why do cells appear distorted? | Too much pressure on the cover slip can crush the cells. Air bubbles or improper mounting medium can also distort the image. |
| M: Why does the image darken when switching to a higher objective? | The light intensity may be too low for higher magnifications. Adjusting the diaphragm or increasing the lamp brightness usually fixes this. |
| M: Too little light | Increase the light intensity on the microscope. Open or adjust the diaphragm/iris to allow more light through. Check and properly position the condenser for optimal illumination. |
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