Study Guide Exam 2 Lab PDF

UNIT LAB 2 LAB 6 – BACTERIA INOCULATION, TRANSFER AND ISOLATION: 1. 4 types of media used in lab: a. Liquid broth – ideal for growing large quantities of bacteria. It’s also useful for observing bacterial growth characteristics in liquid, such as turbidity (cloudiness), which indicat...

UNIT LAB 2 LAB 6 – BACTERIA INOCULATION, TRANSFER AND ISOLATION: 1. 4 types of media used in lab: a. Liquid broth – ideal for growing large quantities of bacteria. It’s also useful for observing bacterial growth characteristics in liquid, such as turbidity (cloudiness), which indicates bacterial density. ▪ Inoculate by transferring bacteria using sterile loop. b. Agar slant – used for maintaining bacterial cultured for longer periods. The slanted surface provides a larger area for growth and helps with oxygen exposure for aerobic organism. ▪ Inoculate by streaking the slanted surface using a sterile loop c. Agar deep – used to test bacterial motility and oxygen requirements. Bacteria that can swim will spread away from the point of inoculation, while non motile bacteria will remain near stab line. ▪ Inoculate by stabbing the agar with sterile needle to introduce bacteria deep into the medium. d. Agar plate – provides large surface are to isolate individual colonies of bacteria, useful for identifying bacterial species and performing colony counts. ▪ Inoculate by streaking the surface of the agar in a specific pattern, such as quadrant streak to isolate individual colonies. 2. What is a colony? And CFU? Colony – a visible mass of microorganisms originating from a single parent cell representing a clone of genetically identical organisms. Colony forming unit (CFU) – a unit to estimate the number of viable bacteria or fungal cells in a sample. It indicates that each colony on an agar plate arose from a single cell or group of cells. 3. Why do we want to isolate colonies of bacteria? Explain the process of quadrant streak. Why isolate colonies? – isolating colonies allows for the identification and study of individual bacterial species. It is important for obtaining pure cultures free from contamination. Quadrant streak process – the loop is used to spread bacteria over four sections of an agar plate in a pattern that progressively dilutes the bacteria. This helps separate individual cells, allowing isolated colonies to form. 4. How can one differentiate/identify bacterial and fungal colonies on an agar plate? Bacterial colonies – generally appear smooth, shiny, and have defined edges. They can be various colors and sizes but tend to be smaller. Fungal colonies – often look fuzzy or cotton-like due to hyphae growth. They are typically larger and have a textured appearance. 5. How can one visually identify different bacterial species on an agar plate? Colony morphology – different species can be distinguished based on colony shape, size, color, texture, elevation, and margin. ▪ Example – some bacteria produce pigments while others may form distinct shapes such as circular, irregular, or filamentous colonies. 6. Explain the process of proper inoculation, incubation, and storage of bacterial plates: Inoculation – inoculate the agar plate using a sterile loop, needle, or swab, depending on the sample and type of inoculation needed. If using the streak plate method, follow a pattern to gradually spread the bacteria across the agar surface to achieve isolated colonies. ▪ Always work near a flame or in a sterile environment to avoid contamination and make sure the inoculating instrument is sterilized (flamed) before and after use. Incubation – place the inoculated agar plate in an incubator at the appropriate temperature to promote bacterial growth. ▪ Incubation time may vary but typically ranges from 24 to 48 hours. Storage – once colonies are visible, store the plates at 4℃ to slow down bacterial growth and preserve the cultures for future analysis. 7. Why do we store inoculated plates agar side up? To prevent condensation from dripping onto the agar surface. Condensation can spread bacteria and cause contamination or smearing making it difficult to observe isolated colonies. 8. Define pure culture, mixed culture and streak for isolation:  Pure culture – a culture containing only one species of microorganism free from contamination by other organisms. Mixed culture – a culture that contains more than one species of microorganism Streak for isolation – a technique used to separate individual bacteria from a mixed culture allowing for the growth of isolated colonies that can be used to obtain a pure culture. LM 8-11 METABOLIC TESTS: 1. Lactose fermentation test – determines if the organism can ferment lactose to produce acid. ▪ Bacteria capable of fermenting lactose produce lactic acid, lowering the pH of the medium. The test often uses a pH indicator which turns yellow in an acidic environment. ▪ Indicator: o Positive: yellow (acid production) o Negative: remains red or turns pink (alkaline) 2. Sucrose fermentation test – assesses the ability of bacteria to ferment sucrose. ▪ Similar to the lactose fermentation test, bacteria that ferment sucrose produce acid, lowering pH ▪ Indicator: o Positive: yellow (acidic) o Negative: no color change or pink (alkaline) 3. Starch hydrolysis test – checks if the bacteria produce the enzyme amylase which breaks down starch. ▪ After incubation, iodine is added to the medium. Iodine binds to starch, turning it blue-black. If the starch has been hydrolyzed by amylase a clear zone appears around the bacterial growth ▪ Indicator: o Positive: clear zone around the growth after iodine application o Negative: blue-black color persists around the growth 4. Indole test – determines if the bacteria can produce indole from the amino acid tryptophan using the enzyme tryptophanase ▪ After incubation, Kovac’s reagent is added. If indole is present it reacts with the reagent to produce a red color. ▪ Indicator: o Positive: red color on the surface o Negative: no color change or yellow 5. Urease test – detects the presence of the enzyme urease which hydrolyzes urea into ammonia and cardon dioxide ▪ The production of ammonia raises the pH causing pH indicator to turn bright pink ▪ Indicator: o Positive: pink color (alkaline) o Negative: no color change (remains yellow or orange) 6. Oxidative-fermentative (OF) glucose test – determines if bacteria metabolize glucose oxidatively or fermentatively. ▪ Two tubes are used – one sealed with oil to create an anaerobic environment and one left open for an aerobic environment. A pH indicator changes based on acid production. ▪ Indicator: o Oxidative: only the open tube turns yellow o Fermentative: both tubes turn yellow o Negative: no color change (remains yellow) 7. Nitrate reduction test – checks if the bacteria can reduce nitrate to nitrate or oxygen gas. ▪ After incubation reagents are added to detect nitrite. If no color change occurs, zinc powder is added to confirm whether nitrate was reduced beyond nitrite. ▪ Indicator: o Positive: red color after the addition of reagents (indicate nitrite) or no color change after zinc addition (indicates nitrogen gas) o Negative: red color after adding zinc (indicates nitrate was not reduced).  8. Tributyrin Agar Test – Determines if bacteria can hydrolyze lipids using lipase enzymes. ▪ If the bacteria produce lipase, they break down the lipid tributyrin in the agar, creating a clear zone around the growth. ▪ Indicators: o Positive: Clear zone around the colonies. o Negative: No change; the agar remains opaque. 9. Starch Agar Test – Same as the starch hydrolysis test mentioned earlier. ▪ Iodine reacts with unhydrolyzed starch to form a blue-black complex. If the bacteria have hydrolyzed the starch, a clear zone appears around the colony after iodine is added. ▪ Indicators: o Positive: Clear zone around bacterial growth. o Negative: Blue-black coloration around the growth persists. LM 12 ENZYMES: 1. What’s an enzyme? Catalyst? Activation energy? Enzyme – is a protein that acts as a biological catalyst, speeding up chemical reactions in cells without being consumed in the process. Enzymes are highly specific to the reactions they catalyze. Catalyst – is any substance that increases the rate of chemical reaction without undergoing any permanent change itself. Activation energy – is the minimum energy required for chemical reaction to occur. Enzymes work by lowering the activation energy, making it easier for the reaction to process. 2. Describe the enzyme-substrate complex: Enzyme-substrate forms when the substrate (the molecule upon which the enzyme acts) binds to the enzyme’s active site. This binding causes a conformational change in the enzyme, making it more efficient at catalyzing the reaction. Once the reaction is complete the products are released, and the enzyme is free to catalyze another reaction. 3. What is the enzyme that catalyzes lactose metabolism? What products are made? The enzyme that catalyzes lactose metabolism is lactase. Lactase breaks down lactose into two simpler sugars: glucose and galactose. 4. How do temperature and pH affect the performance of the lactase enzyme? Temperature – enzymes like lactase have an optimal temperature range (around 37℃ for human enzymes). If the temperature is too low enzyme activity slow down because molecular motion decreases. If the temperature is too high, the enzyme can denature losing its structure and functionality. pH – lactase has an optional pH range, usually around pH 6. If the pH deviates too much from this optimal range, the enzyme’s structure can be altered, decreasing its ability to bind to lactose and catalyze the reaction efficiently. LM 14 SERIAL DILUTION / PLATE COUNT: 1. Describe the serial dilution and pour plate procedure: Serial dilution – this technique involves systematically diluting a bacterial culture to reduce the concentration of cells. In each step, a small, measured volume of the culture is added to a new tune containing a fresh diluent reducing the bacterial containing. These dilutions are then plated to allow for colony formation. Pour plate – after performing serial dilutions, a small volume of the diluted sample is mixed with molten agar and poured into a sterile petri dish. Once the agar solidifies, individual bacterial cells will grow into distinct colonies. 2. What is a colony? CFU? CFU/mL? Colony a visible mass of bacterial cells that originated from a single cell or a group of identical cells, growing on a solid medium.  Colony forming unit (CFU) – a single viable bacterium or a group of bacteria that is capable of dividing and forming a colony on an agar plate. CFU/mL? this represents the number of colony-forming units per milliliter of liquid culture which is used to estimate the concentration of bacteria in a sample. 3. What two objects are achieved by serial dilution and plating of those dilutions of an inoculated nutrient broth? 1) Isolation of individual colonies – by diluting the sample, individual bacterial cells are separated, allowing them to form distinct colonies on the agar plate. This is important for isolating pure cultures. 2) Quantification of bacteria – the number of colonies that form can be counted, allowing for the calculation of the original bacterial concentration in the sample. 4. Describe how serial dilution and plate count is used to determine the concentration of initial undiluted culture: a) Dilute the sample – perform serial dilution of the bacterial culture (e.g., 1:10, 1:100. 1:1,000, etc.). b) Plate the dilutions: plate a known volume from each dilution (usually 100𝜇𝐿) onto nutrient agar plates and incubate them to allow colonies to form. c) Count colonies – after incubation, count the number of colonies on the plate that has a countable range (typically 30-300 colonies). d) Calculate CFU/mL – use the colony count from the selected plate, the dilution factor, and the plated volume to calculate the concentration of the original sample using the formula: 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 CFU/m: = 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑝𝑙𝑎𝑡𝑒𝑑 (𝑖𝑛 𝑚𝐿) LAB 15 SELECTIVE AND DIFFERENTIAL MEDIA: 1. Differentiate, compare, and contrast: Selective media, differential media, and selective media: Selective media – these are designed to suppress the growth of unwanted microorganisms and promote the growth of desired ones. They contain agents that inhibit the growth of certain bacteria while allowing others to thrive. Differential media – these are used to distinguish between different types of bacteria based on observable changes in the medium. These changes are caused by differences in bacterial characteristics. Selective and differential media – these combine the properties of both selective and differential media. They suppress the growth of some bacteria while allowing differentiation of others based on their metabolic characteristics. 2. Describe how each agar type is selective, differential or both. Include how it works—what each is selective or differential (or both) for and what expected result look like: 1) Eosin Methylene Blue (EMB) agar: ▪ Selective – EMB is selective for gm- bacteria because the dyes eosin and methylene blue inhibit the growth of gm+ bacteria. ▪ Differential – EMB differentiates between lactose fermenters and non-fermenters. Lactose fermenters produced acid, which reacts with the dyes to form dark purple or black colonies sometimes with a metallic green sheen (such as E. coli). Non-fermenters produce colorless or pale colonies. ▪ Expected results: o Lactose fermenters – dark purple/black colonies o Strong lactose fermenters (e.g., E. coli) – green metallic sheen o Non-fermenters – colorless colonies 2) Mannitol Sal Agar (MSA): ▪ Selective – MSA is selective for halophilic organisms particularly Staphylococcus species due to its high salt concentration (7.5% NaCl). ▪ Differential – it differentiates bacteria that can ferment mannitol. Mannitol fermentation lowers the pH turning the medium yellow due to the pH indicator phenol red. ▪ Expected results: o Mannitol fermenters (e.g., S. aureus) – yellow halo around colonies  o Non-fermenters – growth but no color change (medium remains red/pink). 3) Blood agar: ▪ Selective – blood agar is not a selective medium; it supports the growth of a wide range of organisms including fastidious bacteria ▪ Differential – it is differential based on hemolysis. Different bacteria produce different types of hemolysis that either completely lyse red blood cells (𝛽-hemolysis), partially lyse them (𝛼-hemolysis), or not at all (𝛾-hemolysis). ▪ Expected results: o 𝛽-hemolysis – complete clearing around colonies (clear zone). o 𝛼-hemolysis – partial clearing or greenish discoloration. o 𝛾-hemolysis – no hemolysis (no change around the colonies). 4) Salmonella-Shigella (SS) agar: ▪ Selective – SS agar is selective for Salmonella and Shigella species. It contains bile salts and brilliant green which inhibits the growth of gm+ bacteria and many gm- enteric bacteria. ▪ Differential – it differentiates based on lactose fermentation and hydrogen sulfide (H 2S) production. Lactose fermenters produce pink colonies while non-fermenters remain colorless. H2S producers such as Salmonella form black-centered colonies due to the reaction with iron salts in the medium. ▪ Expected results: o Lactose fermenters – pink colonies o Non-fermenters (e.g., Salmonella, Shigella) – colorless colonies o H2S producers (e.g., Salmonella) – black-centered colonies LM 16 CONTROL OF GROWTH WITH HEAT: 1. How heat damages microbes: Heat works by denaturing proteins and disrupting the cell membranes and enzymes of microbes which are essential for their survival. As proteins unfold, the microbe loses its ability to function leading to cell death. 2. Compare and contrast use and effectiveness of using dry vs moist heat for disinfection: Moist heat Dry heat Examples Boiling, autoclaving, Flaming, baking pasteurization Effectiveness Moist heat is more effective Dry heat is less effective and than dry heat because water requires higher temperature conducts heat better than air for longer periods to achieve and penetrates cells more sterilization efficiently Mechanism It kills microbes by Kills by oxidizing cell coagulating their proteins. components and denaturing For example, autoclaving proteins, but slower than can kill resistant forms like moist heat spores Common uses Sterilizing surgical Sterilizing glassware and instruments and media in metal instruments that might labs be damaged by moisture 3. Describe a structure some bacteria have to help resist extreme temperatures: ▪ Some bacteria form endospores which are highly resistant to extreme temperatures, desiccation, chemicals, and radiation. ▪ Endospores allow the bacteria to survive in harsh environments until conditions become favorable again. 4. Define thermal death rate (TDR), thermal death time (TDT) and decimal reduction time (DRT) ▪ TDR – the rate at which bacteria are killed at a particular temperature  ▪ TDT – the shortest time required to kill microbes in a sample at a specific temperature ▪ DRT or D-value – time required to kill 90% of the microbes at a specific temperature How did we determine the TDT in out lab? ▪ In the lab we used different water temperatures to observe how long it took for bacteria to be completely killed. The setup included a water bath rack at varying temperatures, an oven set at 100℃, and a glass beaker of water on a hot plate. By placing samples of bacteria in each setup and checking at time intervals, we measured how long it took for all bacteria to be killed at each specific temperature allowing us to determine the TDT for each condition. How is DRT used to evaluate the effectiveness of a given heat treatment? ▪ The DRT measures how long it takes to reduce the microbial population by 90% at a specific temperature. By knowing the DRT, we can evaluate how effective the heat treatment is and how long it needs to be applied to ensure that the microbial population LM 17 CONTROL OF GROWTH WITH UV: 1. How does UV light damage cells? Specifically – what type of mutations do they cause? ▪ UV light primarily damages cells by causing mutations in their DNA. Specifically, UV light leads to the formation of thymine dimers – a type of mutation where two adjacent thymine bases on the same strand of DNA bond together disrupting normal DNA replication and transcription. If left unrepaired, these mutations can lead to cell death or malfunction. 2. What is minimum lethal dose? ▪ The minimum lethal dose is the smallest dose of a harmful agent, such as UV light that is sufficient to kill a particular organism or population of cells. In this case it refers to the minimum amount of UV exposure required to kill cells in a sample. 3. How did we determine the UV minimum lethal dose in lab? ▪ In the lab, we exposed two different bacterial species to varying doses of UV light. We used different exposure times to observe the effect of UV light on cell survival. By increasing the exposure times for each bacterial species, we monitored the bacterial growth and determined the minimum lethal dose – the lowest amount of UV exposure needed to kill all the cells in the sample for each species. This helped us understand the sensitivity of each bacterial species to the UV light. 4. What structure might help bacteria resist high doses of UV light? ▪ Some bacteria such as endospore-forming bacteria (ex., Bacillus and Clostridium species) can resist high doses of UV light. Endospores have a tough outer coating and specialized DNA- repair mechanism that allow them to survive extreme conditions including UV radiation. Additionally, some bacteria possess DNA repair enzymes such as photolyases which can repair the thymine dimers caused by UV exposure, helping them recover from DNA damage.