Micro lab flashcards
What are potential hazards in the lab?
Chemical, biological, fire, electrical, and physical hazards.
What are standard safety practices in the lab?
Wearing PPE, proper labeling, safe chemical handling, and waste disposal procedures.
What are personal protective equipment (PPE) used in the lab?
BSL 1: Lab Coat
BSL 2: Gloves and Goggles
How should various materials be disposed of in the lab?
According to their type: biological waste, sharps, and chemicals have specific disposal protocols.
Glass: Brown plastic bin
Plastic: Orange Bucket
Why is handwashing important, and how do you wash hands correctly?
Prevents contamination; wash hands with soap and water for at least 20 seconds.
What are the parts of a microscope and their functions?
(Review further)
Eyepiece: Magnifies image.
Objective Lenses: Different magnifications.
Stage: Holds the slide.
Light Source: Illuminates specimen.
Coarse Focus Knob: Initial focusing.
Fine Focus Knob: Precise focusing.
What are magnification, resolution, and contrast in microscopy?
Magnification enlarges an image, resolution clarifies detail, and contrast differentiates between specimen parts.
How do you calculate total magnification?
Multiply the eyepiece magnification by the objective lens magnification.
What unit is used to express the size of microbial cells?
Micrometers (µm)
What is the purpose/advantage of making a wet mount?
To observe living organisms, motility, and natural cell shape without staining.
How do you make a wet mount?
Make a well with Vaseline/petroleum jelly, place a drop of liquid sample on a slide, cover with a coverslip, and observe under a microscope.
What is the purpose/advantage of the hanging drop technique?
It provides better observation of motility and avoids drying, allowing longer examination.
How do you perform the hanging drop technique?
Place a drop of liquid on a coverslip, invert it over a depression slide, and observe under a microscope.
How can you tell the difference between Brownian motion and true motility?
Brownian motion is random and caused by water molecules, while true motility shows directional movement.
How can you tell the difference between Brownian motion and true motility?
Brownian motion is random and caused by water molecules, while true motility shows directional movement.
Q: Where are bacteria found?
A: Bacteria are found everywhere, including air, soil, water, and living organisms.
Q: Where are bacteria found?
A: Bacteria are found everywhere, including air, soil, water, and living organisms.
Q: What is the definition of a sterile environment?
A: A sterile environment is free of all living microorganisms.
Q: What factors affect bacterial growth?
A: Temperature, pH, oxygen levels, and nutrient availability.
Q: Define agar medium, petri dish, agar plate, and colony.
A: Agar medium: A gel-like substance for growing bacteria.
Petri dish: A shallow, round dish for culture growth.
Agar plate: A petri dish containing agar medium.
Colony: A group of identical bacterial cells from a single parent cell.
Q: What are standard practices for using agar plates to grow bacteria?
A: Incubate plates upside down, use aseptic techniques, and label plates properly.
Q: What are standard practices for using agar plates to grow bacteria?
A: Incubate plates upside down, use aseptic techniques, and label plates properly.
Q: What is the purpose of a pure culture?
A: To isolate and grow a single species of microorganism for study.
Q: What is contamination? What are some sources of contamination?
A: Contamination: The presence of unwanted organisms.
Sources: Airborne particles, improper handling, and unsterilized equipment.
Q: What is contamination? What are some sources of contamination?
A: Contamination: The presence of unwanted organisms.
Sources: Airborne particles, improper handling, and unsterilized equipment.
Q: How is aseptic technique used to prevent contamination?
A: By sterilizing equipment, using proper handling techniques.
Q: What are the different culture media, and their advantages and disadvantages?
A: Liquid media (good for large bacterial cultures), solid media (isolation of colonies), and selective media (promotes specific bacteria growth).
Q: What are inoculating tools, and when are they used?
A: Inoculating loop (used for liquid cultures and agar plates) and inoculating needle (used for deep cultures).
Q: What is a smear, and what is its purpose?
A: A smear is a thin layer of cells on a slide, used for staining and microscopic examination.
Q: What is a smear, and what is its purpose?
A: A smear is a thin layer of cells on a slide, used for staining and microscopic examination.
Q: What is fixing? How can a smear be fixed, and why?
A: Fixing: Preserving cells on a slide.
Smears can be fixed by heat or chemicals. Fixing prevents the cells from washing off during staining.
Q: What is the purpose of staining a smear?
A: To add contrast and visualize bacterial cells under a microscope.
Q: What is the purpose of staining a smear?
A: To add contrast and visualize bacterial cells under a microscope.
Q: What is a simple stain, direct stain, and negative stain?
A: Simple stain: A single dye to color cells.
Direct stain: Colors the cells themselves.
Negative stain: Colors the background, leaving cells unstained.
Q: What are differential stains, and their purpose? Provide examples.
A: Differential stains distinguish between different types of bacteria. Examples include gram-stain and acid-fast stain.
Note: uses 2 different dyes = crystal violet(primary stain) and safranin(counterstain)
Q: What are differential stains, and their purpose? Provide examples.
A: Differential stains distinguish between different types of bacteria. Examples include gram-stain and acid-fast stain.
Q: What is the purpose of gram-staining?
A: To differentiate bacteria into Gram-positive (purple) and Gram-negative (pink) based on cell wall composition.
Q: What are the steps of gram-staining, and the reagents used?
- Crystal violet (primary stain),
- Iodine (mordant),
- Alcohol (decolorizer),
- Safranin (counterstain).
Q: What are common issues with aseptic technique, and how can you tell something went wrong?
A: Contamination may occur if tools aren’t sterilized properly. Contamination is visible as unexpected growth in cultures.
Q: What are common issues with gram-staining, and how can you tell something went wrong?
A: Over-decolorization can result in Gram-positive cells appearing Gram-negative. Uneven staining may indicate improper technique.
Q: What is the purpose of acid-fast staining?
A: The purpose of acid-fast staining is to identify acid-fast organisms, mainly Mycobacterium species (e.g., Mycobacterium tuberculosis), which have waxy cell walls containing mycolic acids that resist conventional staining methods.
Q: What is the purpose of acid-fast staining?
A: Acid-fast staining is used to identify bacteria with waxy, lipid-rich cell walls (e.g., Mycobacterium species) that resist conventional staining methods due to the presence of mycolic acid.
Q: What are the steps of acid-fast staining, including the reagents and the order they are applied?
- Carbolfuchsin (primary stain) is applied to the smear and a small piece of paper towel can be placed on top.
- The smear is heated to allow the dye to penetrate the cell wall.
- The slide is rinsed with acid-alcohol (decolorizer), which removes the stain from non-acid-fast cells.
- Methylene blue (counterstain) is applied to give non-acid-fast cells a blue color.
Q: What is the clinical application of acid-fast staining?
A: It is used primarily to diagnose diseases like tuberculosis and leprosy caused by Mycobacterium species.
Q: What is the purpose of endospore staining?
A: Endospore staining is used to detect the presence of bacterial endospores, which are highly resistant, dormant structures formed by certain bacteria (e.g., Bacillus, Clostridium) under unfavorable conditions.
Q: What is the purpose of endospore staining?
A: Endospore staining is used to detect the presence of bacterial endospores, which are highly resistant, dormant structures formed by certain bacteria (e.g., Bacillus, Clostridium) under unfavorable conditions.
Q: What are the steps of endospore staining, including the reagents and the order they are applied?
- Malachite green (primary stain) is applied to the smear and heated to allow penetration of the spore.
- The slide is rinsed with water to remove excess dye.
- Safranin (counterstain) is applied to stain vegetative cells red, while spores remain green.
Q: What are the steps of endospore staining, including the reagents and the order they are applied?
- Malachite green (primary stain) is applied to the smear and heated to allow penetration of the spore.
- The slide is rinsed with water to remove excess dye.
- Safranin (counterstain) is applied to stain vegetative cells red, while spores remain green.
Q: What is the clinical application of endospore staining?
A: It helps in identifying bacteria that produce endospores, which is important in diagnosing infections caused by Bacillus anthracis (anthrax) and Clostridium species (e.g., tetanus, botulism).
tldr; helps to identify bacteria containing endospores allowing the correct diagnosis of infection in patient.
Q: How does a mycolic acid cell wall function as a virulence factor?
A: The waxy mycolic acid cell wall provides resistance to desiccation, chemicals, and phagocytosis, allowing bacteria to survive in harsh conditions and evade immune responses.
tldr; waxy layer prevents cell from drying out, being eaten by immune cells, being destroyed by the bodies defenses and surviving tough environments
Q: How do capsules function as virulence factors?
A: Capsules prevent phagocytosis by host immune cells, helping bacteria evade immune defenses and enhance their survival and pathogenicity.
Q: How do endospores function as virulence factors?
A: Endospores allow bacteria to survive extreme environmental conditions (e.g., heat, UV, chemicals), making infections more persistent and difficult to treat.
Q: What is a mixed culture? What is a pure culture?
A: A mixed culture contains more than one type of microorganism, while a pure culture contains only one species of microorganism.
Q: What is the purpose of growing a pure culture?
A: The purpose is to study the characteristics, behaviors, and pathogenicity of a single species of microorganism without interference from other species.
Q: What is an isolated colony? What is its utility?
A: An isolated colony is a group of identical microorganisms derived from a single progenitor. It is useful for identifying and studying a specific microorganism.
Q: What is the purpose of the streak plate method?
A: The purpose is to isolate pure colonies of bacteria from a mixed culture by spreading the bacteria over the surface of an agar plate.
Q: What are the advantages and disadvantages of the streak plate method?
• Advantages: Simple, cost-effective, allows isolation of colonies.
• Disadvantages: Requires skill to achieve good isolation, not suitable for quantitative analysis, and contamination is common.
Q: What is the purpose of quantitative dilution of bacteria?
A: To reduce the concentration of bacteria so that individual colonies can be counted, allowing for quantitative analysis of bacterial populations.
Q: How is quantitative dilution of bacteria performed?
A: A series of serial dilutions is made by transferring a small volume of bacterial culture into a diluent, and then spreading the diluted samples onto agar plates.
Q: What is a statistically valid number of colonies on a plate?
A: A valid number is between 30 to 300 colonies on a plate.
Q: What is selective media? Provide an example.
A: Selective media contain ingredients that inhibit the growth of some organisms while allowing others to grow.
Example: Mannitol Salt Agar (MSA), which selects for Staphylococcus species.
Q: What is differential media? Provide an example.
A: Differential media allow the growth of many organisms but distinguish between them based on certain biochemical characteristics.
Example: Blood Agar, which differentiates based on hemolysis.
Q: What is enrichment media?
A: Enrichment media enhance the growth of a particular organism by providing specific nutrients.
Q: What is the purpose of MSA media?
A: To isolate and differentiate Staphylococcus aureus from other bacteria.
Q: Is MSA media selective, differential, or both?
A: MSA is both selective and differential. It is selective for salt-tolerant bacteria and differentiates based on mannitol fermentation.
Q: What is a positive result on MSA media? What is a negative result?
• Positive: Yellow color (indicates mannitol fermentation).
• Negative: No color change (remains red).
Q: What product results in the color change on MSA media?
A: Acid produced from mannitol fermentation causes the pH indicator (phenol red) to turn yellow.
Q: What is the purpose of EMB media?
A: To isolate and differentiate Gram-negative bacteria, particularly Enterobacteriaceae, and to detect lactose fermenters.
Q: Is EMB media selective, differential, or both?
A: EMB is both selective and differential. It selects for Gram-negative bacteria and differentiates based on lactose fermentation.
Q: What are the possible results on EMB media and how are they indicated?
• Strong lactose fermenter: Metallic green sheen (e.g., E. coli).
• Weak lactose fermenter: Pink or purple colonies.
• Non-lactose fermenter: Colorless or translucent colonies.
Q: What product results in the color change on EMB media?
A: The acid produced from lactose fermentation interacts with the dyes (eosin and methylene blue), resulting in a color change.
Bacteria: E. coli and other lactose producing bacteria
Q: What is catabolism? What are carbohydrates? What is starch, and how is it catabolized by bacteria? What is produced?
A: Catabolism is the breakdown of complex molecules to produce energy. Carbohydrates are sugars and starches. Starch is a polysaccharide broken down by bacteria using amylase into simpler sugars like glucose.
Q: How is starch hydrolysis in bacteria tested? What is a positive result? What is a negative result? What causes the positive result?
A: Bacteria are grown on starch agar, and iodine is added.
• Positive result: Clear zone around bacteria (indicates starch breakdown). • Negative result: Blue-black color with no clear zone. • The enzyme amylase causes the positive result by breaking down starch.
Q: What is glycolysis? What are the products of glycolysis?
A: Glycolysis is the process of breaking down glucose into two molecules of pyruvate. The products are 2 ATP, 2 NADH, and 2 pyruvate molecules.
Q: What is the purpose of the O/F test? How is it performed? What are the results, and how are they interpreted?
A: The O/F (Oxidation-Fermentation) test determines if bacteria use oxidative or fermentative metabolism. Two tubes are inoculated: one covered with mineral oil (anaerobic) and one uncovered.
• Yellow indicates acid production (fermentation/oxidation), and green or blue indicates no acid production. • The color change is due to acid production. The indicator is bromothymol blue. Mineral oil creates anaerobic conditions.
Q: What is the purpose of the fermentation test? How is it performed? What are positive and negative results?
A: The test determines if bacteria ferment specific carbohydrates. A tube with a pH indicator and Durham tube is used.
Q: What is the purpose of the MRVP test? How is it performed? What are the results and product that led to the color change?
A: The MRVP test distinguishes bacteria based on their metabolic byproducts.
• Methyl Red (MR) detects mixed acid fermentation (positive: red). • Voges-Proskauer (VP) detects acetoin production (positive: red). • A negative result remains yellow in both tests.
Q: What is the purpose of the citrate test? How is it performed? What are the results and product that led to the color change?
A: The citrate test determines if bacteria can use citrate as their sole carbon source.
• Positive result: Blue color (ammonia produced). • Negative result: Green (no change). • The indicator is bromothymol blue.
Q: What happens if you incubate the fermentation test tube for over 24 hours? Does this affect the interpretation of the results?
A: Incubating for over 24 hours may result in reversion (bacteria use up all the sugars and start using proteins, causing a pH increase). This can lead to false negative results.
TLDR; Protein=Ammonia, Ammonia=Basic
Q: What are proteins? What are the subunits of proteins?
A: Proteins are large, complex molecules made of amino acids. The subunits are amino acids, which contain an amino group, carboxyl group, and a side chain (R group).
Q: What are exoenzymes? Why do bacteria use exoenzymes?
A: Exoenzymes are enzymes secreted by bacteria to break down large molecules (like proteins or starch) outside the cell for easier absorption of nutrients.
Q: How do you test for gelatin hydrolysis? What is a positive result? How do you double-check, and why use agar instead of gelatin?
A: Gelatin hydrolysis is tested by incubating bacteria in gelatin media.
• Positive result: Liquefaction of gelatin. • Double-check by refrigerating to ensure liquefaction is not due to temperature. Agar is used because it stays solid at incubation temperatures, unlike gelatin.
Q: What is the purpose of the urease test? What is a positive result? What causes the color change?
A: The urease test detects urease, an enzyme that breaks down urea into ammonia.
• Positive result: Pink color. • The color change is due to ammonia, which increases pH. The indicator is phenol red.
Q: What happens after larger protein molecules are broken down extracellularly? What are deamination, decarboxylation, and desulfurization?
A: Proteins are broken into amino acids, which are further catabolized by:
• Deamination: Removal of an amino group. • Decarboxylation: Removal of a carboxyl group. • Desulfurization: Removal of sulfur from amino acids.
Q: What is the purpose of the MIO deep? What are the indicators/color changes?
A: MIO deep tests for motility, indole production, and ornithine decarboxylation.
• Positive motility: Diffuse growth. • Positive indole: Red after adding Kovac’s reagent. • Positive ornithine: Purple color.
Q: What is the purpose of the phenylalanine slant? What are the indicators/color changes?
A: It tests for phenylalanine deaminase activity.
• Positive result: Green color after adding ferric chloride.
Q: What is the purpose of the peptone iron deep? What are the indicators/color changes?
A: It detects hydrogen sulfide (H₂S) production.
• Positive result: Black precipitate forms due to H₂S reacting with iron salts.
Q: What is the utility of differentiating/identifying bacteria based on biochemical tests?
A: Biochemical tests allow the identification of bacteria based on their metabolic properties, which is crucial for diagnosing infections and determining appropriate treatments.
Q: What is the utility of differentiating/identifying bacteria based on biochemical tests?
A: Biochemical tests allow the identification of bacteria based on their metabolic properties, which is crucial for diagnosing infections and determining appropriate treatments.
