Study Guide for Micro Lab Practical Exam #2

If the transformation was not successful, describe the growth on all three types of the plates

+pGLO / LB + Amp🔹 Purpose: Shows that transformation was successful — cells have the plasmid and survive ampicillin resistance because their is no arabinose to induce pGLO.🔹 Expected result: Growth, no glow. -pGLO / LB + Amp🔹 Purpose: Ampicillin kills bacteria unless they have the ampicillin resistance gene, which is part of the pGLO plasmid. Since transformation failed, the bacteria don't have that resistance.🔹 Expected result: No growth, no glow -pGLO / LB🔹 Purpose: there's no GFP gene or arabinose.🔹 Expected result: Normal growth (no resistance or glow).

For colony counting the number of colonies (CFU) should not be less than __ or greater than __

- 30-300 Less than 30 CFU → Not statistically reliable Random error becomes more significant More than 300 CFU → Colonies may merge (confluent growth) Hard to count accurately Overcrowding can inhibit growth of some colonies

What is a primer for PCR?

A primer for PCR (Polymerase Chain Reaction) is a short, single-stranded piece of DNA that is complementary to a specific sequence at the 3' end of the target DNA region. Types of Primers: Forward Primer: Binds to the complementary sequence on the 3' end of the target strand. It serves as the starting point for the synthesis of the new strand in the 5' to 3' direction. Reverse Primer: Binds to the complementary sequence on the opposite strand of DNA, on the 3' end. It facilitates the synthesis of the new strand in the opposite direction (3' to 5').

What is a zone of inhibition? How is it formed?

A zone of inhibition is the clear area around an antibiotic disc on an agar plate where bacteria have not grown. How It's Formed: A lawn of bacteria is spread evenly across the agar plate. A paper disc soaked with an antibiotic (or antimicrobial agent) is placed on the surface. As the plate incubates, the antibiotic diffuses outward from the disc into the agar. If the bacteria are sensitive to the antibiotic, they won't grow near the disc. This results in a clear circular zone—the zone of inhibition—around the disc where bacterial growth is suppressed or killed.

What are the two products of Fermentation? What is a positive tube for the MPN test?

Acid (yellow) and Gas - 💧 What is a Positive Tube for the MPN Test? In the Most Probable Number (MPN) test (used to detect coliforms in water), a positive tube must show: ✅ Turbidity (growth)✅ Gas production in the Durham tube✅ (Optional) Acid production (medium may turn yellow)

what are antibiotics?

Antibiotics are chemical substances used to kill or inhibit the growth of bacteria. They are not effective against viruses, only bacteria. Antibiotics target specific parts of bacterial cells, such as: Cell wall synthesis (e.g., penicillin) Protein synthesis (e.g., tetracycline) DNA/RNA synthesis (e.g., ciprofloxacin) Metabolic pathways (e.g., sulfa drugs)

What is the difference between Asymptomatic and Pre-symptomatic carriers of Covid-19?

Asymptomatic Carriers: Definition: These individuals are infected with SARS-CoV-2 but never develop symptoms of COVID-19, even though they are infected with the virus. Pre-symptomatic Carriers: Definition: These individuals are infected with SARS-CoV-2 and will eventually develop symptoms, but they are not yet showing symptoms at the time of testing

Be able to identify the type of organism (Bacteria, Mold, Yeast) by the Appearance of different colonies, Be able to identify the type of Bacteria by the Color of different colonies and their scientific names

Bacteria: Typically have smooth, glossy, or dry colonies, and their colors can vary from white to yellow, red, or green. Molds: Have larger, more fuzzy or powdery colonies, and colors range widely, including black, green, or white. Yeasts: Have smooth, shiny colonies that are often white, cream, or slightly tan.

What is the difference between cumulative and individual dilution concentrations?

Cumulative Dilution: - Total dilution from the original sample - Multiply all previous individual dilutions - 10⁻¹ × 10⁻¹ × 10⁻¹ = 10⁻³ Individual Dilution: - Dilution of a single step - volume transferred / (volume transferred + diluent) - 1 mL into 9 mL = 10⁻¹

Why and Which size DNA molecules move slower or faster in the gel of Electrophoresis?

DNA moves through a gel matrix (usually agarose) like it's squeezing through a net. Smaller pieces slip through the holes in the gel more easily, while larger pieces get tangled and slowed down. Smaller DNA fragments 👉 move faster and travel farther down the gel. Larger DNA fragments 👉 move slower and stay closer to the wells.

What electrode does DNA move to in Electrophoresis?

DNA moves toward the positive electrode (anode) during electrophoresis. This is because DNA has a negative charge due to its phosphate backbone, and opposite charges attract—so when an electric current is applied, DNA migrates through the gel toward the positive (+) end. 🔋 Quick summary: DNA charge: ➖ Negative Moves toward: ➕ Positive electrode (anode)

What is DNA sequencing?

DNA sequencing is the process of determining the exact order of nucleotides (the building blocks) that make up a DNA molecule. It allows scientists to read the genetic code of an organism or virus, helping to understand its genetic makeup.

What kind of electrophoresis does DNA sequencing use?

DNA sequencing typically uses capillary electrophoresis for the separation of DNA fragments. - It provides high-resolution separation of DNA fragments, making it ideal for sequencing small fragments, such as those generated in Sanger sequencing

What is the difference between direct and indirect methods of enumerating bacteria?

Direct: Count individual cells (e.g., standard plate count, microscope count) Indirect: Estimate based on by-products or turbidity (e.g., OD, spectrophotometer)

What is the difference in color between EMB and Endo plates?

EMB Deep purple, sometimes with a greenish tint Contains eosin Y and methylene blue dyes Endo Light pink to rose-colored Contains basic fuchsin and sodium sulfite

What are the majority of Nucleic Acid Tests use for SARS-CoV-2?

RT-PCR (Reverse Transcription Polymerase Chain Reaction) Description: This is the gold standard for SARS-CoV-2 detection. RT-PCR tests detect the presence of SARS-CoV-2 RNA by first converting the viral RNA into complementary DNA (cDNA) through reverse transcription, followed by amplification of specific regions of the cDNA using PCR. How It Works: RNA from the patient sample (e.g., nasopharyngeal swab) is extracted. The RNA is reverse-transcribed into cDNA. Specific viral gene sequences (such as the spike protein or N-gene) are amplified, and the presence of these amplified sequences indicates the presence of SARS-CoV-2. Fluorescent signals are generated during amplification and detected, providing results. Purpose: RT-PCR is used for confirming current infections by detecting viral RNA. Advantages: High sensitivity and specificity. Highly accurate, considered the gold standard for SARS-CoV-2 detection. Disadvantages: Requires specialized laboratory equipment and skilled personnel. Typically takes several hours to produce results. May require batch processing, leading to delays.

What does RT-PCR stand for?

RT-PCR stands for Reverse Transcription Polymerase Chain Reaction

What is Reverse Transcription?

Reverse Transcription is the process of converting RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. It is essentially the reverse of the normal transcription process, which converts DNA into RNA.

What are the differences between SARS-CoV-2 and COVID-19?

SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2 The virus itself — the pathogen that causes the disease. COVID-19Coronavirus Disease 2019The illness/disease caused by SARS-CoV-2.

How are SARS-CoV-2 viruses obtained for testing?

SARS-CoV-2 viruses (or samples containing SARS-CoV-2) are obtained for testing primarily from patient samples through diagnostic procedures like nasopharyngeal swabs, oropharyngeal swabs, and saliva samples. These samples are collected from individuals who may be showing symptoms of COVID-19 or who have been exposed to suspected cases.

Gram stain results

Gram stain: Gram-positive bacteria: Purple (retains the crystal violet stain due to a thick peptidoglycan layer in the cell wall). Gram-negative bacteria: Pink or red (after decolorization, they retain the counterstain, safranin, because they have a thinner peptidoglycan layer and an outer membrane).

Medical Microbiology: What agar Media was used?

SBA - Sheep Blood Agar Type: Enrichment & Differential Purpose: Detects hemolysis patterns (β, α, γ) used to identify Streptococcus species. Color: Bright red MSA - Mannitol Salt Agar Type: Selective & Differential Purpose: Selects for Staphylococcus (due to high salt); differentiates S. aureus (ferments mannitol, turns yellow) from others. Color: Pink/red base, turns yellow with acid

Sheep blood agar (SBA): how is it an enrichment medium and a differential medium (2 ways)

SBA as an Enrichment Medium Enrichment medium: It supports the growth of fastidious organisms—bacteria that require specific nutrients to grow. The 5% sheep blood provides extra nutrients (like iron and growth factors) that help support the growth of picky pathogens such as Streptococcus and Neisseria species. SBA as a Differential Medium (in 2 ways) 1) Hemolysis Patterns: SBA helps differentiate bacteria based on their hemolytic activity—how they lyse red blood cells. 2) Growth Differences Among Bacteria: Some bacteria grow more robustly on SBA than others, so colony size, morphology, and pigment can help in preliminary identification. There are three types of hemolysis you can observe:

Mannitol salt agar (MSA): how is it selective and/or differential? What property of MSA agar makes it selective?

How is MSA Selective and/or Differential? 🔹 Selective: MSA contains 7.5% sodium chloride (NaCl)—a high salt concentration. This inhibits most bacteria except halotolerant (salt-tolerant) organisms, like Staphylococcus species. 🔹 Differential: MSA contains mannitol (a sugar alcohol) and phenol red (a pH indicator). It differentiates between mannitol fermenters and non-fermenters.

How do you make a lawn of bacteria? What is its purpose?

How to Make a Lawn of Bacteria: - Inoculate broth culture: Start with a dense, actively growing bacterial culture. - Soak a sterile swab in the culture (or use a pipette to spread liquid directly). - Spread the bacteria evenly over the surface of the agar plate: Use the swab to streak back and forth in one direction. Rotate the plate 90° and repeat. Repeat one more time to fully cover the surface. - Let the plate dry (lid on but slightly ajar) for a few minutes before incubating.

What and why are four agar plates used for this lab?

In order to compare the experiments and figue out what allows things to grow, grow, etc.

What was measured with the ruler and what units are used?

In the Kirby-Bauer antibiotic sensitivity test, you measure the zone of inhibition with a ruler, and the units used are millimeters (mm). - You measure the diameter of the zone of inhibition. - a zone of 18 mm might be considered sensitive for one antibiotic but resistant for another, depending on the chart.

What is Long Covid-19?

Long COVID-19, also referred to as Post-Acute Sequelae of SARS-CoV-2 infection (PASC), is a condition in which individuals continue to experience symptoms or develop new health problems weeks or months after their initial recovery from a COVID-19 infection. This occurs regardless of whether the person had mild, moderate, or severe COVID-19 symptoms initially. Long COVID can affect people of all ages, including those who had asymptomatic or mild cases of the virus.

What is needed for Heat-Shock artificial transformation?

Materials Needed: Competent Cells E. coli that have been made chemically competent by treating with cold calcium chloride (CaCl₂) or another divalent cation (like Mg²⁺). This makes the cell membrane more permeable to DNA. Plasmid DNA Circular DNA (e.g. a plasmid with an antibiotic resistance gene or a reporter gene like GFP). Calcium Chloride Solution (CaCl₂) Cold solution used to prepare competent cells. It neutralizes the negative charges on the DNA and the bacterial membrane. Ice Bath Keeps cells cold and stabilizes membranes before and after heat shock. Water Bath or Heat Block (42°C) Used to apply a brief heat shock (~30-60 seconds), which creates a thermal imbalance across the membrane and helps the plasmid enter the cell. LB Broth (Luria Broth) Nutrient-rich media for cells to recover and express the plasmid genes (especially antibiotic resistance) after transformation. Agar Plates with Antibiotic Selective media to grow only the successfully transformed cells. Pipette!! 🔁 Steps Overview: Mix plasmid DNA with competent cells on ice. Incubate on ice (~15 minutes). Heat shock at 42°C for 30-60 seconds. Return to ice for a few minutes. Add LB broth and incubate at 37°C for recovery (~1 hour). Plate on agar with the appropriate antibiotic. Incubate overnight and check for colonies.

Mold

Molds tend to have larger, fuzzy, and more complex colony structures compared to bacteria. Shape: Mold colonies are often fuzzy or woolly in appearance. Size: Molds can be several centimeters in diameter. Color: They often have a range of colors, from white to green, black, gray, or yellow, due to spore production. Texture: Molds are often powdery or velvety with a woolly appearance. Elevation: Mold colonies can be flat or raised, often with irregular edges. Examples of Common Molds: Aspergillus: Green, fluffy, raised colonies with a powdery texture. Penicillium: Blue or green, fuzzy colonies with a characteristic appearance. Rhizopus: White, cottony colonies with black sporangia (spore structures) on top.

What are the advantages and disadvantages of multi-test (like API 20E strip) systems for bacterial identification?

Multi-test systems like the API 20E strip are highly efficient (one strip requirement), cost-effective, and convenient tools for bacterial identification, especially in clinical and microbiological laboratories. However, their use is limited to pure cultures and known bacterial species, and they may not always provide accurate identification for more rare or complex bacteria..

What are the first five steps for a Nucleic Acid Test for SARS-CoV-2 using RT-PCR?

Nucleic Acid Test Steps for SARS-CoV-2 using RT-PCR :• 1. Obtain SARS-CoV-2 Virus from infected patients by Nasopharyngeal Swab Technique• 2. Isolate RNA : Extraction and Purification of RNA from Virus by Spin Columns• 3. Convert Virus RNA into cDNA by Reverse Transcription (RT) • 4. Amplification of cDNA by PCR then Purify by Spin Columns, • 5. Measure the size of PCR fragment by electrophoresis to see if virus was present in sample, check controls• 6. Use DNA Sequencing Machine to determine the sequence • 7. Sequencing Analysis

What are Nucleic Acid Tests and Nucleic Acid Amplification Test?

Nucleic Acid Tests (NATs): Nucleic Acid Tests refer to a broad category of tests that are used to detect the presence of specific nucleic acids (DNA or RNA) in a sample. These tests help identify pathogens (like bacteria, viruses, or fungi), genetic mutations, or even trace amounts of DNA in clinical samples. Nucleic Acid Amplification Tests (NAATs): NAATs are a subset of NATs that specifically focus on amplifying the nucleic acids in a sample to increase their concentration, making it easier to detect even small amounts of target genetic material.

Microscope objective and ocular lens

Objective Lenses Primary lenses that magnify the specimen. Four lenses with varying magnifying powers. 4x, 10x, 40x, 100x Ocular Lens Eyepiece lens for viewing specimens.

What does PFU stand for and what is a phage plaque?

PFU stands for Plaque Forming Unit. - A PFU is a measure of infectious virus particles, specifically how many bacteriophages are capable of infecting and lysing (bursting) bacterial cells to form plaques on an agar plate. - 1 PFU = 1 infectious virus particle that led to the formation of a plaque. It's similar to CFU (colony-forming unit) for bacteria, but used for viruses. - A plaque is a clear zone on a bacterial lawn (solid growth of bacteria on agar) caused by virus-induced cell lysis. - It forms when a phage infects a bacterial cell, replicates, and bursts the cell. - New phages released from that cell go on to infect nearby cells. - This creates a clear circle or zone of inhibition where the bacteria have been killed.

What is a phage plaque, and how do you determine titer of a viral stock?

Phage Plaque: Cloudy area is the lawn of the bacteria; plaques are the clear zones formed due to lysis by bacteriophage (kill zone) PFU/Ml=Number of plaques/DXV

What is PCR and how does PCR work?

Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify small segments of DNA, generating millions of copies from a very small initial sample. This amplification allows for the analysis of DNA in greater detail 1) Denaturation (94-98°C): DNA is heated to separate the double strands into two single strands. 2) Annealing (50-65°C): The temperature is lowered, allowing primers to bind to their complementary sequences on the single-stranded DNA. 3) Extension (72°C): Taq polymerase adds nucleotides to the primers to synthesize new DNA strands, extending the sequence. 4) Repeat: Steps 1-3 are repeated for 30-40 cycles, doubling the DNA each time. 5) Final Elongation: After the last cycle, a final extension ensures any remaining single-stranded DNA is fully extended.

What are the positive and negative controls for gel electrophoresis?

Positive control: A sample that is known to contain the target DNA to ensure the PCR worked correctly. Negative control: A sample that doesn't contain the target DNA to check for contamination.

What sugar is used in the Presumptive & Completed test? What agars were used in the Confirmed test?

Presumptive Test: Sugar used: The lactose sugar is used in the Presumptive test. This test checks for the presence of coliform bacteria, which ferment lactose and produce gas as a byproduct. If gas is produced in the lactose broth, it suggests the possible presence of coliforms. Completed Test: Sugar used: Similar to the Presumptive test, lactose is also used in the Completed test to check for gas production and acid formation. This confirms the presence of coliform bacteria (including E. coli) that ferment lactose. Confirmed Test: In the Confirmed test, agar media are used to identify and confirm the presence of coliforms or fecal coliforms, and the following agars are commonly used: Eosin Methylene Blue (EMB) Agar: This selective and differential agar is used to isolate coliform bacteria and differentiate them from non-coliforms. Coliforms typically produce dark colonies with a characteristic metallic green sheen on EMB agar. This is especially useful for confirming the presence of fecal coliforms. MacConkey Agar: This is another selective and differential agar used to isolate gram-negative bacteria, such as coliforms. Coliforms on MacConkey agar typically appear as pink colonies due to lactose fermentation, which produces acid and changes the pH indicator in the medium.

What is a PCR fragment made of?

Primers: Short single-stranded DNA sequences that bind to the target DNA on either side of the region to be amplified. They serve as starting points for DNA synthesis. Target DNA: The specific DNA sequence you want to amplify. The PCR process copies this segment. Nucleotides: The building blocks (adenine, thymine, cytosine, and guanine) that are added by the DNA polymerase to build the new DNA strand during the extension step. DNA polymerase: An enzyme (usually Taq polymerase) that synthesizes the new DNA strands by adding nucleotides to the primers. Amplified DNA: The result of the PCR process, which is a large number of copies of the target DNA fragment.

Can the API strip be used with a pure or mixed culture? Explain why.

Pure Culture: The API strip should be used with a pure culture. This is because the test relies on identifying specific biochemical reactions from a single type of bacteria. In a mixed culture, different bacteria may have varying biochemical properties, which would interfere with the accuracy of the results, making it impossible to attribute a specific reaction to one bacterial species. A pure culture ensures that the reactions observed in each test correspond to a single species. Mixed Culture: The API strip cannot be used with a mixed culture. If multiple bacterial species are present, the results will be unclear because different species may give different results in the same test wells. This would lead to inconclusive or misleading results, as the biochemical reactions would not be representative of a single bacterial species.

What is the purpose of the antibiotic lab?

Purpose of a Bacterial Lawn: ✅ Antibiotic testing: So you can clearly see zones of inhibition around antibiotic discs. ✅ Phage experiments: Helps visualize plaques (clear spots) caused by viral lysis. ✅ Uniform background: Ensures any changes (like glowing colonies or dead zones) are easy to see. ✅ Consistent growth: Good for quantitative studies and creating a standard environment.

For each of the media used to identify your unknown you should know: (1) what a positive and negative result looks like (2) the basics of the biochemical reaction occurring in a positive test and (3) if applicable, the enzyme involved in each test,

SIM Test (Sulfur, Indole, Motility) Positive result: Black precipitate for H₂S production Red ring for indole production Diffuse growth (cloudiness) for motility Negative result: No black precipitate No color change for indole No diffusion of growth (non-motile) Biochemical reaction: Tests for sulfur reduction, tryptophan metabolism (indole), and bacterial motility. Enzyme: Tryptophanase, cysteine desulfurase.

What are the advantages and disadvantages of the standard plate count when compared to other methods in the lab?

SPC (Standard Plate Count) ✅ Counts only viable cells (living bacteria) ✅ Inexpensive & easy ❌ Time-consuming (24+ hrs) ❌ Cannot count anaerobes well or clumped cells Other Methods: ✅ Faster methods like turbidity (OD) via spectrophotometer ❌ Spectrophotometer is expensiveeeee ❌ Other methods may count dead cells (e.g., direct microscope count) ❌ Takes time for colonies to grow ❌ Indirect methods don't confirm viability

How are countable plate numbers different between Bacteriophage or SPC?

SPC (bacteria) Colonies 30-300 CFU CFU/mL - Fewer than 30 colonies are statistically unreliable, and more than 300 makes it hard to distinguish and count individual colonies because they can overlap or merge. Bacteriophage assay Plaques 30-300 PFU (or ≤250) PFU/mL - Similar concept, but instead of colonies, you are counting clear zones called plaques formed by viruses killing host bacteria. - But in some protocols, the upper limit may be 250 plaques (depending on plate size and the phage used), because plaques spread differently than bacterial colonies.

Note: page 152 in the lab manual has the incorrect answer for the example calculation due to not applying the amount of mlused on the plate, make sure you know how to calculate CFU/ml. (2.3x10^9 CFU/ml)

Scientific notation is fine for answers of dilution problems

How is EMB selective and differential? Know the appearance of E. coli on EMB agar.

Selective: EMB agar inhibits Gram-positive bacteria, allowing only Gram-negative bacteria to grow. Differential: EMB agar differentiates bacteria based on their ability to ferment lactose. Lactose fermenters, like E. coli, produce green colonies, while non-lactose fermenters produce colorless colonies. E. coli's distinctive green sheen on EMB agar is a key identifying feature.

How is Endo selective and differential? Know the appearance of E. coli on Endo agar

Selective: Endo agar inhibits the growth of Gram-positive bacteria and selects for Gram-negative bacteria, especially members of Enterobacteriaceae. Differential: It differentiates bacteria based on lactose fermentation. Lactose fermenters like E. coli produce red colonies with a metallic sheen, while non-lactose fermenters produce colorless colonies. E. coli's distinctive red colonies with a metallic sheen on Endo agar make it easily identifiable.

Review all the types of agar plates used, are they: Selective, Differential, Enrichment, Appearance?

Sheep Blood Agar (SBA) Selective: No Differential: Yes (hemolysis) Enrichment: Yes Appearance: Red agar with zones of hemolysis (greenish or clear) Mannitol Salt Agar (MSA) Selective: Yes Differential: Yes (mannitol fermentation) Enrichment: NO Appearance: Red agar (yellow around mannitol fermenters) Eosin Methylene Blue (EMB) Selective: yes Differential: yes (lactose fermentation) Enrichment: No Appearance: Purple or greenish sheen for lactose fermenters Nutrient Agar Selective: No Differential: No Enrichment: No Appearance: light beige or clear Sabouraud Dextrose Agar (SDA): 5.6pH fungi Selective: Yes Differential: No Enrichment: No Appearance: Yellowish (supports fungal growth)

What are all the tests used for the presumptive, confirmatory and completed tests?

Summary of Tests: Presumptive Test: Lactose broth with Durham tube: Gas production suggests coliforms. Multiple tubes method: Statistical estimation of coliform density. Confirmed Test: EMB agar: Coliforms produce dark or metallic green colonies. MacConkey agar: Lactose fermenters produce pink colonies. Completed Test: Lactose broth with Durham tube: Gas production confirms lactose fermentation. Gram stain: Gram-negative, rod-shaped bacteria confirm coliforms. Indole test (optional): Positive for E. coli.

Why do we need to test for Water Contamination?

1. Public Health Protection: 2. Water Quality Monitoring 3. Regulatory Compliance: Legal Standards: Governments and health organizations set guidelines for safe drinking water, such as the EPA (Environmental Protection Agency) standards in the United States. Testing ensures that water suppliers comply with these regulations, preventing contamination levels from exceeding safe limits. 4. Agriculture and Industrial Uses: 5. Environmental Protection: 6. Early Detection of Problems: 7. Consumer Confidence:

Gel Electrophoresis: What is Electrophoresis? How does Electrophoresis work? What is the charge on DNA

- Electrophoresis is a laboratory technique used to separate DNA, RNA, or proteins based on their size and charge by applying an electric current to a gel matrix. ⚡ How Does Gel Electrophoresis Work? Gel Preparation:A gel (usually made of agarose for DNA) is poured into a tray with wells at one end to load samples. Loading DNA Samples:DNA is mixed with loading dye (for visibility and density) and pipetted into the wells. Electric Current Applied:Electrodes are attached to both ends of the gel—negative (-) at the top, positive (+) at the bottom. DNA Movement:Since DNA is negatively charged due to its phosphate backbone, it migrates toward the positive electrode. Separation by Size: Smaller DNA fragments move faster and farther through the gel. Larger fragments move slower and stay closer to the wells. Visualization:A dye like ethidium bromide or GelRed binds to DNA and fluoresces under UV light, revealing the band patterns. DNA has a negative charge because of the phosphate groups in its backbone.

What are the five characteristics of Coliform bacteria? (there are 5 of them)

- Gram-negative: Coliform bacteria are Gram-negative, meaning they do not retain the crystal violet stain in the Gram staining procedure and instead take on a pink color due to their thinner peptidoglycan cell walls. - Rod-shaped (bacillus) - Facultative anaerobes - Lactose fermenters: A key feature of coliform bacteria is their ability to ferment lactose, producing acid and gas as byproducts. - Presence in the intestines of warm-blooded animals

Phenol red: pH indicator - used in many of the labs this semester; what color is it when it is acidic?

- In acidic conditions (pH below 7), phenol red turns yellow. - In neutral or slightly basic conditions (pH around 7), phenol red is red. - In strongly basic conditions (pH above 7), phenol red turns pink.

how to evaluate the effectiveness of antibiotics and sensitivity of a bacterium - Sensitive, Intermediate, Resistant

- Measure the diameter (in mm) of each zone. - Compare it to a standard chart provided by the Clinical and Laboratory Standards Institute (CLSI). Sensitive (S): the antibiotic is effective — bacteria are clearly inhibited. Intermediate (I): The antibiotic may work, but effectiveness is borderline — higher dose or combination may be needed. Resistant (R): The antibiotic is not effective — bacteria continue to grow.

What are the 5 characteristics of a good Fecal indicator organism?

- Present in feces of warm-blooded animals - Not pathogenic - Survives longer than pathogens in the environment - Easy to detect and quantify - Present in the same environmental conditions as pathogens

Why test water for E. coli and not specific pathogens?

- Specific to the intestines of warm-blooded animals - Abundant - Not typically harmful in small numbers - Testing for E. coli is a practical, cost-effective, and reliable method for determining water safety. It serves as a proxy indicator for fecal contamination and the potential presence of harmful pathogens, making it more feasible than testing for each specific pathogen individually. By detecting E. coli, we can infer the likelihood of pathogens in water and take appropriate action to prevent waterborne diseases.

Most Probable Number (MPN) Table 44.1 on p. 186 - know how to use table; What are the units?

- The Most Probable Number (MPN) method is a statistical technique used to estimate the number of viable microorganisms (often coliforms) in a liquid sample, such as water. - The MPN is usually expressed as the number of most probable organisms per 100 milliliters (MPN/100 mL). This unit reflects the concentration of microorganisms in the sample. Steps for Using the MPN Table: Prepare a series of tubes with different dilutions of the water sample, typically in three different sets or dilutions (e.g., undiluted, 1:10, and 1:100). Inoculate the tubes with the water sample and incubate them. Examine the tubes for signs of microbial growth (e.g., turbidity or gas production). Record the results as positive (growth) or negative (no growth) for each dilution. For example, you might have: 3 tubes at the undiluted level 3 tubes at the 1:10 dilution 3 tubes at the 1:100 dilution Match the pattern of positive and negative results to the MPN table. The table will typically show the expected MPN for different combinations of positive and negative results across the dilution levels.

What sugar is used to control the araC gene regulation and how does it work?

- The sugar used to control araC gene regulation is arabinose. - Arabinose sugar binds to the AraC protein. - This changes the shape of AraC, so it can no longer hold the DNA in a loop. - RNA polymerase can now bind to the promoter. 👉 Result: Transcription occurs, and genes like GFP are turned on.

Why are viruses called obligate intracellular parasites?

- They must infect a host cell to replicate - Once inside, they hijack the host's cellular machinery to produce new viral particles. - They cannot reproduce on their own - Viruses lack the machinery (like ribosomes or enzymes for metabolism) needed to grow and divide.

What is the objective of the enumeration of bacteria lab?

- To quantify the number of viable bacteria in a sample using serial dilutions and the standard plate count (SPC) method. -Learn how to calculate CFU/mL (colony-forming units per milliliter).

What are the five ways Covid-19 is WORSE than the Flu?

- higher transmission rate - longer incubation period - higher risk of complications - high mortality rate - long term effects

What are the five organ systems and their parts that SARS-CoV-2 directly infects?

- respiratory system - nervous system - cardiovascular system - gastrointestinal system - renal (urinary) system

Microscope objective and ocular lens

-Objective Lenses: Primary lenses that magnify the specimen. Four lenses with varying magnifying powers. 4x, 10x, 40x, 100x -Ocular Lens: Eyepiece lens for viewing specimens.

Why is E. coli a good choice for a microbe in this experiment?

1) Fast Growing E. coli has a short generation time (about 20 minutes under ideal conditions), so colonies form quickly—often overnight. 2) Easy to Culture It grows well on basic, inexpensive media like nutrient agar or LB agar. 3) Visible Colonies E. coli forms distinct, easy-to-count colonies, which is perfect for colony-forming unit (CFU) calculations. 4) Well Studied It's one of the most well-known bacteria, so scientists have a deep understanding of its growth patterns and behavior. 5) Safe Strains Are Available The lab uses non-pathogenic strains (like E. coli K-12), which are safe for student labs. 6) Indicator Organism It's often used in water testing and food safety because its presence can indicate fecal contamination.

Kirby-Bauer method: how is it set up and run? What did we do differently from the original set up?

1) Prepare the Mueller-Hinton Agar Plate: Use standardized Mueller-Hinton agar, which supports consistent diffusion of antibiotics. 2) Create a Bacterial Lawn: Dip a sterile swab into a standardized bacterial suspension (usually 0.5 McFarland turbidity). Swab the entire plate in three directions, turning the plate 60° each time, to ensure even coverage (a lawn of bacteria). 3) Place Antibiotic Disks: Use sterile forceps or a disk dispenser to place paper disks impregnated with known concentrations of antibiotics onto the agar surface. Press each disk gently to ensure contact with the agar. 4) Incubate the Plate: Place the plate in a 35-37°C incubator for 16-24 hours. Incubate the plate upside down to prevent condensation from dripping onto the agar. 5) Measure Zones of Inhibition: Use a ruler or caliper to measure the diameter (in millimeters) of the clear zones where bacteria failed to grow. - Used Mueller-Hinton agar, your lab may have used a general nutrient agar - Applied disks with dispenser or sterile forceps

What are the different ways to enumerate bacteria?

1) Standard Plate Count (SPC) 2) Phage Plaque Assay - direct method that counts lytic phage determined by a dilution technique 3) Turbidity / Optical Density (OD) using spectrophotometer 4) Direct microscopic count 5) Filtration 6) Most Probable Number (MPN)

What was the purpose of the Bacteriophage lab?

1) 🔬 Understand Viral Infection and Replication in Bacteria - The lab shows how viruses require a host cell to replicate—illustrating why they are called obligate intracellular parasites. 2) Learn How to Perform a Plaque Assay - As phages infect and lyse bacteria, they create clear zones called plaques. 3) 📏 Quantify Virus Concentration Using PFU/mL - By counting plaques and factoring in the dilution, you can calculate the plaque-forming units per milliliter (PFU/mL) in the original sample.

How do you Evaluate Health Information on the Internet?

1)Check the Source 2)Assess the Domain: 3)Evaluate the Content: 4)Look for Quality Indicators 5)Beware of Bias

Aseptic technique

1. General Principles of Aseptic Technique Sterilize Tools: Always sterilize tools like loops, needles, and forceps by passing them through a flame (Bunsen burner) or using an autoclave for larger equipment. Work in a Clean Area: Work in a clean, uncluttered environment. Ideally, perform microbiological work in a designated lab area with a laminar flow hood or Bunsen burner to create an updraft of sterile air. Minimize Exposure: Minimize the time that plates, tubes, or petri dishes are open to air to prevent airborne contaminants from entering. Wear Protective Gear: Always wear gloves, a lab coat, and safety glasses to protect yourself and the culture. 2. Handling Petri Plates (Agar Plates) Label Before Use: Label the underside of the agar plate with necessary information Keep Plates Closed: . Sterilize Tools Between Transfers: Avoid Cross-contamination 3. Handling Tubes with Agar Flame the Tube Opening: Before and after transferring a sample into or from a tube, flame the neck of the tube briefly. Seal the Tubes Properly: reseal the tube tightly to prevent contamination. Avoid touching the rim 4. Handling Broth Cultures Inoculation Process: sterilize it by flaming Incubation: After inoculating a broth culture, securely close the cap to minimize contamination. Observe for Contamination: If a broth shows unusual turbidity (cloudiness) or a change in appearance, it may indicate contamination 5. Disposal and Cleanup Proper Disposal of Cultures: Always dispose of used culture plates, tubes, and other materials in designated biohazard waste containers. Clean Equipment: Disinfect work surfaces and tools before and after use.

What are the two types of operons and how are they different?

1. Inducible Operons - Default state: OFF - Turned ON by: Presence of a specific molecule (inducer) - Function: Often control catabolic pathways (breaking things down) 🔹 Example: The lac operon It stays off unless lactose (the inducer) is present. When lactose is around, it binds the repressor and allows the operon to be transcribed. 2. Repressible Operons - Default state: ON - Turned OFF by: A corepressor binding to the repressor protein - Function: Often control anabolic pathways (building molecules) 🔹 Example: The trp operon It produces enzymes to make tryptophan. When tryptophan is present in high amounts, it acts as a corepressor, binding the repressor and shutting the operon off.

What indicators were used in class?

1. Phenol Red Used in: Fermentation tests, including sugar fermentation. Color change: Yellow when acidic (pH < 6.8). Red at neutral pH (around 7.0). Pink when alkaline (pH > 7.4). Purpose: Indicates acid production from sugar fermentation by bacteria, which results in a pH drop. 2. Methyl Red Used in: Methyl Red-Voges-Proskauer (MR-VP) test. Color change: Red when the pH is acidic (pH < 4.4). Yellow at neutral or alkaline pH. Purpose: Used to detect mixed acid fermentation. A positive result (red color) indicates that the organism produces stable acids. 3. Bromothymol Blue Used in: Used in media to detect the presence of CO₂, such as in Simmon's Citrate agar or in tests of bacterial respiratory activity. Color change: Yellow in acidic conditions (pH < 6.0). Blue in alkaline conditions (pH > 7.6). Purpose: Detects acid production from bacterial growth or gas production due to bacterial metabolism. 4. Neutral Red Used in: It can be used in selective media like MacConkey agar for the detection of lactose fermentation. Color change: Red in acidic environments (pH < 6.8). Colorless at neutral or alkaline pH. Purpose: Identifies lactose fermenters in media, where fermentation results in acid production and the appearance of red colonies.

How long does SARS-CoV-2 stay viable on various surfaces?

1. Plastic and Stainless Steel Duration: 2-3 days (48-72 hours) Reason: These non-porous surfaces can keep the virus viable longer than porous ones. The virus can remain infectious for up to three days, though the viral load decreases over time. 2. Cardboard Duration: 24 hours Reason: The virus can survive for a day on cardboard, but it doesn't remain infectious for as long as on plastic or stainless steel. After a day, it's significantly less viable. 3. Copper Duration: 4 hours Reason: Copper surfaces are considered to have antiviral properties. SARS-CoV-2 doesn't remain viable as long on copper, likely due to the material's ability to destroy the virus more rapidly. 4. Glass Duration: Up to 4 days Reason: Like plastic and stainless steel, glass is a non-porous material, so the virus can survive longer than on porous surfaces. 5. Fabric (Cloth) Duration: Up to 2 days Reason: The virus can survive for a short time on fabric, but it is generally much less stable due to the porous nature of textiles. 6. Paper (e.g., tissues, napkins) Duration: Up to 3 hours Reason: Similar to cardboard, paper is a porous material that doesn't allow the virus to stay viable as long. The virus tends to lose infectivity relatively quickly on paper.

What are the damages to each organ system and their parts caused by SARS-CoV-2 infection?

1. Respiratory System Parts affected: Nose, throat, trachea, bronchi, and especially the lungs (alveoli) Effects: Pneumonia, acute respiratory distress syndrome (ARDS), coughing, shortness of breath 2. Nervous System Parts affected: Brain, cranial nerves (especially olfactory nerve), spinal cord Effects: Loss of smell (anosmia), headaches, brain fog, strokes, seizures, encephalitis 3. ❤️ Cardiovascular System Parts affected: Heart muscle (myocardium), blood vessels, endothelial cells Effects: Myocarditis, arrhythmias, blood clots, stroke, heart attacks 4. Gastrointestinal System Parts affected: Esophagus, stomach, intestines (especially small intestine), liver Effects: Diarrhea, nausea, vomiting, liver enzyme elevation 5. Renal (Urinary) System Parts affected: Kidneys (especially proximal tubule cells), bladder Effects: Acute kidney injury, proteinuria, increased risk of kidney failure

What are the three types of gene transfer used by a organisms?

1. Transformation Definition: Uptake of naked DNA fragments from the environment. How it works: A bacterium takes in free DNA (usually from a dead cell) and incorporates it into its own genome. Example: Streptococcus pneumoniae can gain antibiotic resistance genes this way 2. Transduction Definition: DNA is transferred from one bacterium to another by a bacteriophage (a virus that infects bacteria). How it works: A phage accidentally picks up bacterial DNA during replication and transfers it to another bacterial cell. Types: Generalized transduction: Any gene can be transferred. Specialized transduction: Only certain genes are transferred. 3. Conjugation Definition: Direct transfer of DNA between two bacterial cells through a physical connection called a pilus (or mating bridge). How it works: A donor cell with an F plasmid (fertility plasmid) transfers genetic material to a recipient cell. Example: Spread of antibiotic resistance via plasmids.

Which age group has the most cases of COVID-19 in the USA?

65+

How is a virus's lytic cycle different from the lysogenic cycle?

: Lytic Cycle: This is the "active" and destructive cycle. 🔹 Steps: Attachment - Virus binds to the surface of a host cell. Entry - The viral DNA or RNA is injected into the host. Replication - The host cell's machinery replicates the viral genome and makes viral proteins. Assembly - New viruses are assembled from the replicated parts. Lysis - The host cell bursts (lyses), releasing the newly formed viruses to infect other cells. ✅ Key Features: Fast process. Host cell is destroyed. Produces many viruses quickly. Causes acute infections (e.g., flu, COVID-19, common cold). Lysogenic Cycle: This is the "hidden" or dormant phase. 🔹 Steps: Attachment and Entry - Same as lytic. Integration - Viral DNA integrates into the host's chromosome and becomes a prophage. Replication with Host - Every time the host cell divides, it copies the viral DNA too. Trigger into Lytic Cycle - Certain conditions (e.g., stress, UV light) can trigger the virus to switch to the lytic cycle. ✅ Key Features: Longer and dormant phase. Host cell is not immediately destroyed. Virus stays hidden for a while. Causes latent infections (e.g., herpes, HIV in early stages). Quick Analogy: Lytic = Robber breaks in, ransacks the place, and blows it up. Lysogenic = Spy hides in the house, copies info every day, and waits for a signal to strike.

What is a Candle jar and its purpose?

A Candle Jar is a simple and classic device used in microbiology to create a low-oxygen, high-carbon dioxide (CO₂) environment - 🎯 Purpose: To support the growth of bacteria that don't grow well in normal atmospheric oxygen levels, such as: Streptococcus species Haemophilus influenzae

What is a Durham Fermentation Tube (DFT)? What constitutes a positive DFT?

A Durham Fermentation Tube (DFT) is a microbiological test used to detect carbohydrate fermentation and gas production by microorganisms 🔬 What is a Durham Fermentation Tube? It's a test tube containing: Broth medium with a specific sugar (like glucose, lactose, or sucrose). pH indicator (usually phenol red). A small, inverted Durham tube (a little test tube placed upside down inside the big one) to catch gas bubbles.

What is a Bacteriophage?

A bacteriophage (often just called a phage) is a virus that infects bacteria

Bacteria: concept of clones and colonies - one cell or a small group of cells produces a colony

A clone is a population of identical cells derived from a single parent cell. All cells in the clone are genetically identical, meaning they carry the same genetic material. In bacteria, a clone results from asexual reproduction (typically binary fission), where a single bacterium divides into two genetically identical daughter cells. A colony is a visible mass of bacterial cells that all originate from a single parent cell or a small group of genetically identical cells. When bacteria are plated on agar media, they grow in clusters, and each colony originates from a single cell or a small group of cells.

What is a countable plate for SPC?

A countable plate for SPC is any plate that has a count between 30 and 300 for bacteria viruses are 25-250

Know how to use the graph of absorbance (Optical Density) vs. concentration.

A graph of OD (y-axis) vs. concentration (x-axis, usually CFU/mL) shows a linear relationship within a certain range. It helps you estimate the number of bacteria in a sample by measuring how much light is absorbed by the culture. - As bacterial concentration increases, the absorbance increases. - This method does not distinguish between live and dead cells — it just measures turbidity (cloudiness). - How to use the graph: 1. Measure OD of your bacterial sample using a spectrophotometer. 2. Find that OD value on the y-axis of the graph. 3. Use the graph to trace horizontally to the line and then drop down vertically to find the CFU/mL.

Does a positive Presumptive MPN test mean that the water is absolutely unsafe to drink, explain why?

A positive Presumptive MPN test does not absolutely confirm that the water is unsafe to drink — but it is a strong warning sign What does a positive Presumptive MPN test mean? It indicates that lactose-fermenting, gas-producing bacteria (like coliforms) are likely present in the water sample. Coliforms are indicator organisms, meaning their presence suggests possible fecal contamination. ❗ But does it guarantee pathogens are in the water? No. The presumptive test detects bacteria that may be coliforms, but: Some non-coliform bacteria can give false positives. It doesn't identify specific harmful pathogens like Salmonella or Shigella.

Bacteria

Bacterial colonies can have a wide range of appearances based on the species. Here are some general features: Shape: Bacterial colonies can be circular, irregular, punctiform (small), or filamentous. Size: Typically from 1 mm to several millimeters. Color: Bacterial colonies often range from white to cream, yellow, red, or even green. The color can also indicate the production of pigments. Texture: Can be smooth, rough, or dry. Some may be shiny, while others appear dull. Edge: Edges may be entire, lobed, or irregular. Elevation: Bacteria may have flat, raised, convex, or umbonate (central raised) colonies. Examples of Common Bacterial Colonies: Escherichia coli (E. coli): Cream or off-white, smooth, round colonies. Staphylococcus aureus: Golden-yellow colonies, smooth and moist. Pseudomonas aeruginosa: Greenish-blue, smooth, and moist colonies, often with a fruity odor. Streptococcus pyogenes: Small, translucent, grayish colonies, often with hemolysis on blood agar.

Do bacteriophages infect Eukaryotes or Prokaryotes?

Bacteriophages infect only prokaryotes, specifically bacteria. They do not infect eukaryotic cells (like those of humans, animals, or plants). That's why they're called bacteria-phages — they specifically target bacteria.

What is a CFU? Why is CFU/ml used rather than bacteria/ml? What is the least number of cells to form a colony?

CFU is a Colony Forming Unit - It represents one viable (living) bacterial cell—or a group of cells—that can multiply and form a visible colony on an agar plate - We use CFU/mL to count only the bacteria capable of forming colonies—not dead ones or ones that can't divide. - CFU/mL gives a more accurate measurement of viable (living) bacteria that can grow and divide. - only one viable cell is required to form a colony

How is Covid-19 mainly transmitted? What are the main symptoms?

COVID-19 is mainly transmitted through respiratory droplets and aerosols when an infected person: Talks, Coughs, Sneezes, Breathes heavily Symptoms: Fever or chills loss of taste Cough Shortness of breath or difficulty breathing Fatigue Muscle or body aches Loss of taste or smell Sore throat Headache Congestion or runny nose Nausea or vomiting Diarrhea

What are two methods for artificial transformation?

Chemical Transformation: Calcium Chloride + Heat Shock How it works: Cells are treated with cold calcium chloride (CaCl₂) to make their membranes more permeable. Plasmid DNA is added. A brief heat shock (usually 42°C for ~30-60 seconds) creates a thermal imbalance that allows the DNA to enter the cell. Used with: E. coli and other bacteria. Why it's effective: Calcium neutralizes the negative charges on the DNA and cell membrane. Electroporation: How it works: Cells and DNA are mixed and exposed to a short electric pulse. The electric shock creates tiny pores in the cell membrane through which DNA can enter. Used with: Bacteria, yeast, and eveterm-38n mammalian cells. Why it's effective: It doesn't rely on chemicals and works on a wide range of organisms.

How to read a pipette accurately

Choose the correct pipette for the volume you need. Ensure the pipette tip is free of air bubbles and properly filled. For adjustable micropipettes: Set the desired volume on the display or dial. Read the liquid level at eye level: For manual pipettes, read the lowest point of the meniscus (for concave liquids). For micropipettes, read the volume on the digital display. Align your eye level with the meniscus to avoid parallax errors. Use proper technique: Pre-wet the tip, avoid air bubbles, and pipette steadily. Record the volume once it's measured.

Review bacterial shapes and arrangements

Cocci: Spherical; arrangements include pairs, chains, clusters, and tetrads. Bacilli: Rod-shaped; arrangements include singles, pairs, chains, and palisades. Spirilla and Spirochetes: Spiral-shaped; tend to be single cells, with spirochetes being more flexible.

Which plates should have growth and which no growth and why? THIS COULD BE WRONG

Control: +pGLO / LB + Amp🔹 Purpose: Shows that transformation was successful — cells have the plasmid and survive ampicillin resistance because their is no arabinose to induce pGLO.🔹 Expected result: Growth, no glow. +pGLO / LB + Amp + Arabinose🔹 Purpose: Shows that GFP is expressed only when arabinose is present.🔹 Expected result: Growth and glowing colonies. Non Control: -pGLO / LB + Amp🔹 Purpose: Shows that untransformed cells can't grow in ampicillin.🔹 Expected result: No growth. -pGLO / LB🔹 Purpose: Positive control to show cells are alive and healthy.🔹 Expected result: Normal growth (no resistance or glow). __________________________________ ✅ So, the plates with pGLO are controls because: They validate transformation success. They test the plasmid's function (resistance, fluorescence). They compare transformed vs. untransformed cells under different conditions. D IS THE ONLY ONE THAT GLOWS

Define: Coronaviruses, Zoonotic Disease, Pandemic, Pneumonia, Ventilator, Infected Respiratory Droplets?

Coronaviruses Definition:A large family of viruses that can cause illnesses ranging from the common cold to more severe diseases such as SARS (Severe Acute Respiratory Syndrome), MERS (Middle East Respiratory Syndrome), and COVID-19. They are named for the crown-like spikes on their surfaces. Zoonotic Disease Definition:A disease that spreads from animals to humans. Many viruses, including some coronaviruses, are zoonotic in origin. Examples include COVID-19, Ebola, and Rabies. Pandemic Definition:A global outbreak of a disease that affects a large number of people across multiple countries or continents. COVID-19 was declared a pandemic by the World Health Organization (WHO) in March 2020. Pneumonia Definition:An infection of the lungs that causes the air sacs (alveoli) to fill with fluid or pus. It leads to coughing, fever, and difficulty breathing. COVID-19 can cause viral pneumonia in severe cases. Ventilator Definition:A medical device that helps a person breathe when they are unable to do so on their own. It pushes air (and oxygen) into the lungs and is often used for patients with severe respiratory distress, such as those with critical COVID-19. Infected Respiratory Droplets Definition:Tiny drops of fluid from the nose or mouth that contain viruses or bacteria and are expelled when an infected person coughs, sneezes, talks, or breathes heavily. These droplets are the main way COVID-19 spreads from person to person.

From the information generated by the strip, be able to calculate digit profile number and use a flowchart to identify your particular unknown bacteria

Each well on the API 20E strip tests for a specific biochemical characteristic of the bacteria (e.g., sugar fermentation, enzyme activity, etc.). After incubating the strip, you will get positive or negative results, which are recorded as either a "1" (positive) or "0" (negative). Positive result ("1"): Indicates that the bacteria show a characteristic (e.g., fermentation or enzyme activity) that matches the expected reaction. Negative result ("0"): Indicates that the bacteria do not show the expected characteristic. You start by writing down each digit for each test (1 or 0). Then, write the test results as a series of digits, like 1010010101. This digit profile number is used to match against a database or flowchart to identify the unknown bacterium. - Once you have the digit profile, you can use a flowchart (or an API database) to identify the bacterium Flowchart Example: Step 1: Is the result for Test 1 (e.g., sugar fermentation) positive? Yes → Proceed to Step 2 No → Identify as Group A or Group B (based on negative results) Step 2: Is the result for Test 2 (e.g., enzyme activity) positive? Yes → Proceed to Step 3 No → Identify as Group C or Group D Continue this pattern, progressively narrowing down the identification based on the test results. The last step will provide you with the species name or genus that matches the digit profile.

Endospore stain protocols

Endospore Staining Protocol: Prepare the Slide: Prepare a bacterial smear and heat-fix it to the slide. Use an Endospore Stain kit (usually includes malachite green and safranin). Staining Process: Malachite Green (Primary Stain): Apply malachite green to the smear and steam it over boiling water for about 5 minutes. The heat helps the malachite green penetrate the tough spore coat. Rinse with water: After steaming, rinse the slide with water to remove excess malachite green. Safranin (Counterstain): Apply safranin for 1 minute, rinse with water, and blot dry. Resulting Colors: Endospores: Retain the green color of malachite green. Vegetative cells: Take on the red color of safranin.

Endospore stain results

Endospore: Endospores: Green (stained with malachite green, which penetrates the tough spore coat). Vegetative cells: Red (stained with safranin, which stains the non-spore part of the cell).

How do we distinguish E. coli from Enterobacter aerogenes?

Escherichia coli (E. coli) and Enterobacter aerogenes are both Gram-negative bacteria commonly found in the intestines of humans and other animals, and they are members of the family Enterobacteriaceae 1. Indole Test E. coli: Positive for the indole test. This means that E. coli can break down tryptophan to produce indole, which reacts with Kovac's reagent to produce a red or pink color. Enterobacter aerogenes: Negative for the indole test. Enterobacter aerogenes does not produce indole when tryptophan is metabolized. Lactose Fermentation (MacConkey Agar) E. coli: Positive for lactose fermentation. On MacConkey agar, E. coli produces pink colonies because it ferments lactose and produces acid. Enterobacter aerogenes: Positive for lactose fermentation as well, but the colonies may appear light pink or off-white with a less intense color compared to E. coli

What is used for extraction and purification of RNA from Virus?

Extraction and Purification of RNA from Virus by• Spin Binding Columns•!!!! Lysis Virus, RNA binds to beads in column, wash away other parts of virus, elute concentrated purified RNA • Spins done in microcentrifuge • Can be done in less than 30 min - RNA binds to beads

How are genes regulated?

Genes are regulated through gene expression control, which allows cells to turn specific genes on or off depending on their needs. This regulation ensures that proteins are made only when needed. Used heavily in Eukaryotes. In Prokaryotes (e.g., E. coli): Most gene regulation occurs at the transcription level (when mRNA is made from DNA). 1. Operon Model An operon is a group of genes under the control of a single promoter. Inducible Operons (like lac operon): OFF by default. Turned ON in the presence of a specific substance (e.g., lactose). Example: The lac operon is activated when lactose is present and repressor is removed. Repressible Operons (like trp operon): ON by default. Turned OFF when the end product is present in excess (feedback inhibition). Example: The trp operon is repressed when tryptophan is abundant. 2. Regulatory Proteins: Repressors bind to operators to block transcription. Activators enhance the binding of RNA polymerase to the promoter to increase transcription. In Eukaryotes: Gene regulation is more complex and can occur at multiple levels: 1. Chromatin Structure: Tightly packed DNA (heterochromatin) = genes are OFF. Loosely packed DNA (euchromatin) = genes are ON. 2. Transcription Factors: Proteins that help RNA polymerase bind to DNA. Can activate or repress transcription. 3. Epigenetic Modifications: DNA methylation (adds methyl groups): silences genes. Histone acetylation (adds acetyl groups): activates genes. 4. Post-transcriptional Control: mRNA splicing, editing, transport, and degradation. 5. Translational & Post-translational Control: Control of when and how proteins are made or activated.

Gram Stain protocols

Gram Staining Protocol: Prepare the Slide: Prepare a bacterial smear on a clean microscope slide and allow it to air dry. Heat-fix the smear by gently passing the slide through the flame of a Bunsen burner. Staining Process: Crystal Violet (Primary Stain): Apply crystal violet to the smear for 1 minute, then rinse with water. This stains all cells. Iodine Solution (Mordant): Apply iodine solution for 1 minute, then rinse with water. Iodine forms a complex with the crystal violet, helping the dye adhere to the cell wall. Decolorization (Alcohol or Acetone): Add alcohol or acetone for 10-20 seconds, then rinse with water. This step differentiates the cells: Gram-positive cells retain the crystal violet-iodine complex. Gram-negative cells lose the crystal violet-iodine complex. Safranin (Counterstain): Apply safranin for 1 minute, then rinse with water and blot dry. This stains the decolorized Gram-negative cells. Resulting Colors: Gram-positive cells: Retain the purple color of crystal violet. Gram-negative cells: Take up the red/pink color of safranin.

Can Asymptomatic SARS-CoV-2 infected people get any injures from the virus?

Silent Organ Damage: Even if not symptomatic, the virus can cause subclinical damage to organs such as the lungs, heart, kidneys, and liver. This damage might not manifest as overt symptoms but could have long-term consequences. For instance, some studies suggest that the virus can still cause inflammation in the lungs, potentially leading to long-term respiratory problems or reduced lung function. Blood Clotting Issues: SARS-CoV-2 has been linked to increased blood clot formation, which can lead to issues like deep vein thrombosis (DVT), pulmonary embolism, or even strokes. These clotting events might occur without obvious symptoms in asymptomatic carriers. Potential Long-Term Effects ("Long COVID"): Some asymptomatic individuals may develop Long COVID, a condition in which symptoms persist for weeks or months after the initial infection. These lingering effects can include fatigue, brain fog, heart palpitations, muscle weakness, and other issues, even though the individual was asymptomatic during the acute phase of the illness. The exact mechanisms of Long COVID are still being studied, but it suggests that even if the initial infection is mild or asymptomatic, the body can still experience prolonged issues. Possible Cardiovascular Impacts: The virus has been shown to have an effect on the heart, even in asymptomatic individuals. This can include myocarditis (inflammation of the heart muscle), which can potentially lead to long-term cardiovascular complications, especially in young, otherwise healthy people. Immune System Effects: The virus may trigger immune responses that could affect the body in subtle ways. For example, immune cells may become activated even in asymptomatic individuals, potentially leading to autoimmune responses or increased inflammation that can affect various body systems.

What is Silent Spreading of Covid-19

Silent spreading of COVID-19 refers to the asymptomatic or pre-symptomatic transmission of the virus. In these cases, individuals who are infected with SARS-CoV-2 do not show symptoms or show symptoms later but can still transmit the virus to others. - It leads to undetected spread in the community, especially in crowded settings like workplaces, schools, and social gatherings.

There is a lot of overlap between this lab and the SPC lab, how are they the same and how are they different?

Similarities: 1) Serial Dilutions - Both labs use serial dilution techniques to reduce concentration to a countable level. 2) Agar Plates - Both rely on solid agar media to visualize microbial activity. 3) Counting Units - Both measure growth using countable plates: CFU (Colony Forming Units) for SPC and PFU (Plaque Forming Units) for phages. 4) Quantitively results: - The goal in both is to calculate the concentration of microbes/viruses in the original sample. 5) Lawn Bacteria: - A uniform lawn is required in both—SPC for colony isolation and phage lab for plaque visibility. 6) Dilutions: - To reach a plate with 30-300 (or 30-300+) colonies or plaques for accurate quantification. Differences 1) SPC lab Measures viable bacterial cells. while Bacteriophage lab measures infectious viral particles. 2) SPC lab has end result: Colonies (visible bacterial growth). while bacteriophage has end result: Plaques (clear zones from cell lysis). 3) SPC Unit: CFU/mL (Colony Forming Units/mL). while bacteriophage unit: PFU/mL (Plaque Forming Units/mL). 4) SPC has Growth = multiplication of bacteria themselves. while bacteriophages have plaques form as a result of viral infection and lysis of bacteria. 5) SPC doesn't involve a virus while bacteriophages involve bacteriophages (viruses that infect bacteria). 6) SPC uses nutrient agar while bacteriophage uses soft agar overlay technique.,

*Why are some wells under or over filled?

Some need more air to grow properly, some need absolutely no air to grow properly

What are the distinguishing features of colony growth for Staphylococcus vs. Streptococcus?

Staphylococcus spp. Colony Size: Medium to large Appearance: Round, smooth, often raised Color: Creamy, white, or golden-yellow (especially S. aureus) Hemolysis (on SBA): S. aureus: Beta-hemolytic (clear zone around colony) S. epidermidis: Non-hemolytic (gamma) Growth Pattern: Discrete, well-separated colonies Oxygen Requirement: Facultative anaerobe Catalase Test: Positive Streptococcus spp. Colony Size: Small to pinpoint Appearance: Matte, dry, or glistening depending on species Color: Translucent or grayish Hemolysis (on SBA): S. pyogenes: Beta-hemolytic S. pneumoniae: Alpha-hemolytic (greenish zone) Enterococcus or other species: Gamma (no hemolysis) Growth Pattern: Often in chains, colonies may merge Oxygen Requirement: Aerotolerant anaerobe Catalase Test: Negative Quick Tips to Differentiate: Catalase test: Bubbles = Staph (+); No bubbles = Strep (-) Hemolysis type: Can help identify species within each genus Colony size and appearance: Staph = larger, more creamy; Strep = smaller, more translucent

How do you determine the size of a PCR fragment by electrophoresis?

Step-by-Step Guide: 1) Run a DNA ladder (marker) in one lane of the gel: This ladder contains DNA fragments of known sizes (e.g., 100 bp, 500 bp, 1000 bp, etc.). It acts like a ruler for comparison. 2) Load your PCR product into another lane of the same gel. 3) Run the gel using an electric current. DNA (negatively charged) will migrate toward the positive (red) electrode. 4) Stain the gel (commonly with ethidium bromide or GelRed) and view under UV light. 5) Compare the distance your PCR band traveled with the bands from the DNA ladder: Use a ruler or imaging software to measure the migration distance of your PCR product and the ladder bands. Plot these distances (in mm or cm) against the 6) log of the base pair size of each ladder band to make a standard curve (semi-log graph). 7) Estimate the size of your PCR product by locating where it falls on the standard curve.

How is DNA sequencing used to determine the type of SARS-CoV-2 variant?

Summary of How DNA Sequencing Identifies SARS-CoV-2 Variants: 1) RNA from the virus is extracted and converted to cDNA. 2) PCR amplification produces many copies of the viral genome. 3) Next-generation sequencing (NGS) reads the genetic sequence of the virus. 4) Bioinformatics tools compare the sequence to reference genomes to identify specific mutations and classify the SARS-CoV-2 variant. 5) Mutations in the spike protein or other regions help distinguish between different variants. By using DNA sequencing, scientists can track the spread of different variants, monitor for new mutations, and evaluate the impact of these variants on disease outcomes, immunity, and vaccine effectiveness. This helps in making informed decisions for public health responses, such as vaccine updates and public health guidelines.

What are the steps for NAT testing for SARS-CoV-2?

Summary of Steps: 1) Sample collection from a patient (e.g., nasal/throat swab or saliva). 2) RNA extraction from the sample. 3) Reverse transcription (RT) of RNA to cDNA. 4) PCR amplification of target SARS-CoV-2 genes. 5) Real-time detection of amplification, usually with fluorescence. 6) Interpretation of results (positive/negative/indeterminate). 7) Quality control checks to ensure accuracy. 8) Result reporting to healthcare providers.

What is the IMVIC test?

The IMViC test is a series of four biochemical tests used to identify and differentiate Enteric bacteria—especially between Escherichia coli and Enterobacter aerogenes. I - Indole Test Purpose: Tests for the ability to degrade tryptophan into indole. Positive result: Red/pink layer after adding Kovac's reagent. E. coli: Positive M - Methyl Red Test Purpose: Tests for production of stable acids from glucose fermentation. Positive result: Red color after adding methyl red. E. coli: Positive V - Voges-Proskauer Test Purpose: Detects acetoin (a neutral fermentation product). Positive result: Red color after adding VP reagents A and B. Enterobacter aerogenes: Positive C - Citrate Utilization Test Purpose: Tests if the organism can use citrate as the sole carbon source. Positive result: Blue color in Simmon's citrate agar. Enterobacter aerogenes: Positive

SIM medium: Which test does each letter stand for?

The SIM medium is a combination medium used to test for Sulfur (S) reduction, Indole (I) production, and Motility (M). Here's what each letter stands for: S - Sulfur Reduction: This test checks whether the bacterium can reduce sulfur-containing compounds (like cysteine) to produce hydrogen sulfide (H₂S). A positive result is indicated by a black precipitate in the medium. I - Indole Production: This test detects whether the bacterium can break down the amino acid tryptophan to produce indole. After adding Kovac's reagent, a red ring at the top of the medium indicates a positive result. M - Motility: This test determines if the bacterium is motile. If the bacterium is motile, it will spread throughout the medium, causing it to appear cloudy. Non-motile bacteria will only grow along the stab line, leaving the surrounding medium clear. In summary, SIM stands for: S - Sulfur reduction (H₂S production) I - Indole production (from tryptophan) M - Motility (ability to spread in the medium)

What are the controls and why? THIS COULD BE WRONG

The controls are 1) LB/AMP/+pGLO 2) LB/AMP/ARABINOSE/+pGLO Why? They contain +pGLO; they help you verify that your transformation worked and understand the effect of the plasmid under specific conditions. 🌟 What is pGLO? pGLO is a plasmid that contains: GFP gene (green fluorescent protein) — glows under UV light 🌟 bla gene — gives resistance to ampicillin araC gene — regulates the GFP gene; only turns it on when arabinose is present Overall: +pGLO / LB + Amp🔹 Purpose Shows that transformation was successful — cells have the plasmid and survive ampicillin resistance because their is no arabinose to induce pGLO.🔹 Expected result: Growth, no glow. +pGLO / LB + Amp + Arabinose🔹 Purpose: Shows that GFP is expressed only when arabinose is present.🔹 Expected result: Growth and glowing colonies. -pGLO / LB + Amp🔹 Purpose: Shows that untransformed cells can't grow in ampicillin.🔹 Expected result: No growth. -pGLO / LB🔹 Purpose: Positive control to show cells are alive and healthy.🔹 Expected result: Normal growth (no resistance or glow). ✅ So, the plates with pGLO are controls because: They validate transformation success. They test the plasmid's function (resistance, fluorescence). They compare transformed vs. untransformed cells under different conditions.

What type of operon is in the Lab experiment? Justify your answer.

The operon in the lab experiment with the pGLO plasmid is an inducible operon. - The pGLO plasmid contains the araC operon, which is inducible—it turns ON in the presence of arabinose.

*What was the purpose of the Bacteriological Examination of Water?

The purpose of the Bacteriological Examination of Water is to detect and identify harmful microorganisms (especially pathogenic bacteria) that may be present in water, indicating contamination and potential risks to public health. This examination is essential for ensuring that water sources are safe for drinking, recreational use, and other purposes

What was the regulatory or inducible factor?

The regulatory or inducible factor in the pGLO transformation experiment is arabinose (a sugar). WHY: - Arabinose turns on (induces) the GFP (green fluorescent protein) gene in the pGLO plasmid. - The pGLO plasmid places the GFP gene under the control of the arabinose operon promoter (araC-controlled). - When arabinose is present, it activates the promoter, allowing GFP to be transcribed and translated. - As a result, only bacteria grown on plates with arabinose will glow green under UV light.

What was the selective agent? Why was it used?

The selective agent in the pGLO transformation experiment is ampicillin (an antibiotic) WHY: - Selects for transformed bacterial cells that have successfully taken in the pGLO plasmid by killing untransformed ones because: - The pGLO plasmid contains the bla gene, which gives resistance to ampicillin. - Only transformed bacteria (those that received the pGLO plasmid) can grow in the presence of ampicillin. - Untransformed bacteria (no plasmid) cannot survive on ampicillin plates.

Thioglycollate medium: - How does the oxygen gradient affect the growth of aerobes, anaerobes, & facultative anaerobes? - Be able to recognize the type of growth for bacteria in a thioglycollate tube. - Why should you not shake the thioglycollate medium?

Thioglycollate medium is used to determine the oxygen requirements of bacteria by creating an oxygen gradient in the medium. Oxygen concentration is highest at the top of the tube and lowest at the bottom. The medium also contains sodium thioglycollate, which reduces oxygen to create an anaerobic environment. - Oxygen Gradient and Growth Patterns Aerobes: These organisms require oxygen to grow. They will only grow at the top of the thioglycollate medium, where oxygen concentration is highest. Anaerobes: These organisms cannot tolerate oxygen and grow in an anaerobic environment. They will grow at the bottom of the tube, where oxygen concentration is lowest. Facultative Anaerobes: These organisms can grow with or without oxygen. They will grow throughout the medium, but they tend to grow more at the top in the presence of oxygen, as they prefer aerobic respiration when oxygen is available. Recognizing Growth in a Thioglycollate Tube Aerobe Growth: Growth will be confined to the very top of the tube, where oxygen is available. Anaerobe Growth: Growth will be confined to the bottom of the tube, where oxygen is absent. Facultative Anaerobe Growth: Growth will be throughout the medium, with the most growth near the top (where oxygen is present) and less growth toward the bottom. Microaerophile Growth: These bacteria require low levels of oxygen. They will grow just below the surface, in the area where oxygen concentration is lower than atmospheric levels but not completely absent. Shaking the thioglycollate medium would disturb the oxygen gradient that has formed in the tube. The gradient allows for differentiation between aerobic, anaerobic, and facultative bacteria, and shaking would mix the oxygen throughout the medium, making it impossible to accurately assess the bacteria's oxygen requirements and growth patterns.

Know how to use a bacterial identification flow chart for identification

Tips for Using a Flowchart: Know your bacterial sample: Always start with what you know (Gram stain and morphology). Be systematic: Follow each step of the chart, don't skip tests. Understand test results: Make sure you know what each test is looking for and how to interpret results. Keep a record of results: Write down the results of each test for easier reference.

Know how to read and use Table 31.2 on pp. 136-140 (will be provided on exam) to determine the Sensitive, Intermediate or Resistant.

Tips: - Always match zone size to the correct antibiotic row. - Measure precisely edge to edge across the center of the disk. - Use a fresh, clean ruler or caliper for accuracy. - Some labs may give you a custom chart based on the specific bacterial species you're testing.

Define the following terms: transformation, competences, artificial competences, genetic engineering, inducible operons, repressible operons, plasmid

Transformation Definition: The process by which bacteria take up naked DNA (usually plasmids or fragments) from their environment. Example: A bacterium absorbs a gene for antibiotic resistance from its surroundings. 🔓 Competence Definition: A bacterium's natural ability to take up DNA from the environment. Some bacteria are naturally competent, like Streptococcus pneumoniae. ⚡ Artificial Competence Definition: Competence that's induced in the lab using treatments like calcium chloride (CaCl₂) or electroporation to make bacterial cell walls more permeable to DNA. Used often in transformation experiments with E. coli. Genetic Engineering Definition: The intentional modification of an organism's DNA to achieve desired traits (e.g., making insulin, glowing jellyfish genes in plants). Involves tools like plasmids, restriction enzymes, and CRISPR. 🔁 Inducible Operon Definition: A gene system that is usually "off" but can be turned "on" when a specific substance (inducer) is present. Example: lac operon in E. coli—activated when lactose is present. Repressible Operon Definition: A gene system that is usually "on" but can be turned "off" when the end product is abundant. Example: trp operon in E. coli—repressed when tryptophan levels are high. 🔄 Plasmid Definition: A small, circular piece of extra-chromosomal DNA in bacteria that replicates independently. Often used as a vector in genetic engineering to insert new genes into cells.

What are the serious symptoms of COVID-19, where you need to seek immediate medical attention?

Trouble breathing Persistent pain or pressure in the chest New confusion Inability to wake or stay awake Bluish lips or face (or pale/gray skin in darker skin tones) Severe dizziness or fainting Signs of a stroke (slurred speech, one side of face drooping, weakness)

what is the objective of this lab?

Use control plates to analyze experimental results Use positive and negative controls to: - Prove the bacteria are healthy. - Confirm that ampicillin kills non-transformed cells. - Show that GFP expression requires both the plasmid and the sugar arabinose.

How would you diagnose patients using PCR and gel electrophoresis?

Using PCR (Polymerase Chain Reaction) and gel electrophoresis 1. Sample Collection: Clinical samples 2. DNA/RNA Extraction: 3. PCR Amplification: 4. Gel Electrophoresis: 5. Diagnosis: Band Comparison: Positive control: A sample that is known to contain the target DNA to ensure the PCR worked correctly. Negative control: A sample that doesn't contain the target DNA to check for contamination. 6. Interpretation: Positive Result: If a band corresponding to the target sequence appears in the gel, it suggests the presence of the pathogen or genetic sequence associated with the disease. Negative Result: If no band appears, the target sequence was not detected, meaning the pathogen or genetic condition is likely absent.

What is the role of the heat and ice baths in transformation?

❄️ Ice Bath (Cold Step) Stabilizes the membrane: Keeps the bacterial cell membrane rigid so the plasmid DNA stays near the surface of the cell. Prepares cells: After treatment with calcium chloride, the cold keeps the pores open and prevents early uptake. Allows DNA binding: The DNA adheres loosely to the outer surface of the bacterial cell membrane. 🔥 Heat Shock (42°C for ~30-60 sec) Creates thermal imbalance: Rapid change from cold to hot creates a pressure gradient across the membrane. Increases membrane permeability: This shock causes temporary holes to form in the cell membrane, allowing plasmid DNA to enter the cell. Think of it like a "zip" that briefly opens and then closes. 🔁 Why both are important together: Without the cold, the cells wouldn't properly bind the DNA or stay stable. Without the heat, the DNA couldn't pass through the membrane.

What is social distancing? Why is there a need for protective measures? How can you protect yourself from COVID-19?

What is Social Distancing? Definition:Social distancing means keeping a safe space between yourself and other people who are not from your household. The general recommendation is to stay at least 6 feet (2 meters) apart. Purpose:To reduce the spread of infectious diseases—like COVID-19—by limiting close contact where respiratory droplets might be exchanged. Why Is There a Need for Protective Measures? Reasons: COVID-19 spreads easily: It is transmitted through respiratory droplets when an infected person coughs, sneezes, talks, or breathes. Asymptomatic spread: People can spread the virus even if they don't feel sick. To protect vulnerable populations: Including the elderly, immunocompromised, and those with underlying conditions. To prevent overwhelming healthcare systems: Slowing the spread gives hospitals and healthcare workers the ability to care for all patients effectively. How Can You Protect Yourself from COVID-19? ✅ Basic Protective Measures: Wash your hands frequently with soap and water for at least 20 seconds. Use hand sanitizer with at least 60% alcohol if soap and water are not available. Wear a mask in crowded or indoor settings. Avoid touching your face, especially your eyes, nose, and mouth. Maintain social distance from people not in your household. Stay home if you're feeling sick. Clean and disinfect frequently touched surfaces daily. ✅ Additional Steps: Get vaccinated and stay up to date with booster shots. Avoid large gatherings, especially indoors. Improve ventilation in enclosed spaces. Monitor your health and get tested if you have symptoms or were exposed to someone with COVID-19.

Always include ( x 10 8 per ml with bacteria number) for concentration

When reporting bacterial concentrations, you're expressing the number of colony-forming units (CFUs) per milliliter of sample — and that number is usually very large. To make it readable and standardized, you use scientific notation. For example: Instead of writing: 250,000,000 CFU/mL You write: 2.5 × 10⁸ CFU/mL The "× 10⁸ per mL" part refers to: The scale (how big the number is). The units (CFU per mL).

Yeast

Yeast Yeasts form smooth, moist colonies similar to bacteria but are generally larger and may show some texture or smoothness. Shape: Yeast colonies are typically smooth, round, and moist. Size: Yeast colonies tend to be larger than bacterial colonies (about 1-2 mm). Color: They can be white, cream, or off-white, though some species produce colorful colonies (e.g., Candida). Texture: Yeasts are usually smooth or glistening, moist colonies. Elevation: Flat, raised, or convex colonies. Examples of Common Yeasts: Candida albicans: White, creamy, smooth colonies. Saccharomyces cerevisiae: Creamy, round, and smooth colonies. Cryptococcus neoformans: Smooth, round, white or slightly tan colonies, often with a mucous appearance. Colony Color and Scientific Names: Staphylococcus aureus: Yellow to golden colonies (produces pigments). Escherichia coli: Off-white, smooth colonies, often with a shiny appearance. Pseudomonas aeruginosa: Greenish colonies due to the production of pyocyanin pigment. Penicillium species: Blue-green colonies. Aspergillus species: Greenish or grayish colonies with a fuzzy texture.

How does the araC gene regulation work?

araC gene → codes for AraC regulatory protein. - AraC protein: Represses gene expression when no arabinose is present. - Activates gene expression when arabinose is present.. - This change allows RNA polymerase to transcribe the downstream gene — GFP — causing glowing colonies under UV light.

Can Asymptomatic SARS-CoV-2 infected people still spread the virus?

duh...

Spectrophotometer: direct or indirect

indirect method An instrument that measures the proportions of light of different wavelengths absorbed and transmitted by a pigment solution.

How do indicators work in order to identify biochemical reactions?

pH indicators: Detect changes in acidity or alkalinity. Redox indicators: Detect changes in the oxidation-reduction potential. Substrate indicators: Detect breakdown products from bacterial metabolism of substrates. Enzyme substrate indicators: Detect specific enzymatic activity. Gas indicators: Detect the presence of gas production.

Bacterial Transformation Experiment

purpose of this lab is to give non-ampicillin resistant E. coli the plasmid that will encode for a gene that will give E. coli ampicillin resistance *Organism used*: Escherichia coli that does not contain the beta-galactosidase and is not ampicillin resistant is used in this experiment *Plasmid used*: the plasmid pGal is used in this experiment because it encodes for *beta-galactosidase* which cleaves the X-gal plasmid and allows for the expression of the pGal plasmid. This plasmid also contains genes for the production of the enzyme *beta-lactamase* which inactivates ampicillin.

Enumeration of bacteria Phage Plaque Assay

viable colony count, direct cell count

What is a countable plate for Bacteriophage?

✅ 30 to 300 plaques 📌 Why this range? <30 plaques:Too few to be statistically reliable — errors in dilution or plating have a big effect on the count. >300 plaques:Too many plaques may overlap, making it hard to count individual ones accurately — this is called a "confluent lawn."

How do you calculate pfu/ml?

✅ PFU/mL = (Number of Plaques × Dilution Factor) / Volume Plated (in mL) - 🔬 Example: You plate 0.1 mL of a 10⁻⁶ dilution, and count 45 plaques on the agar plate. Step-by-step: Plaques counted = 45 Dilution factor = 10⁶ (because dilution was 10⁻⁶) Volume plated = 0.1 mL PFU/mL = (45 × 10⁶) / 0.1PFU/mL = 4.5 × 10⁸

What is the difference between broad spectrum antibiotic and narrow spectrum antibiotic?

🌍 Broad-Spectrum Antibiotics - Definition: These antibiotics are effective against a wide range of bacteria, including both Gram-positive and Gram-negative organisms. - Examples: Tetracycline Ampicillin Chloramphenicol - When used: When the exact bacterial cause is unknown - For serious infections that might involve multiple types of bacteria - Pros: Useful when you're unsure of the specific pathogen. - Cons: Can disrupt normal microbiota, leading to side effects like yeast infections or antibiotic resistance. 🎯 Narrow-Spectrum Antibiotics - Definition: These antibiotics target specific groups of bacteria—either Gram-positive or Gram-negative, but not both. - Examples: Penicillin G (mostly targets Gram-positive) Vancomycin - When used: When the specific bacterial pathogen is identified - Pros: Less damage to the normal flora, lower chance of antibiotic resistance. - Cons: Not effective if the infection involves multiple or unidentified bacteria.

What bacteria can grow or not grow on MSA? How and which bacteria change the color of the MSA plate?

🎨 How and which bacteria change the color of the MSA plate? The pH indicator, phenol red, turns yellow in acidic conditions. Staphylococcus aureus ferments mannitol → produces acid → lowers pH → turns plate yellow. Staphylococcus epidermidis cannot ferment mannitol → no acid → no color change (plate stays pink/red).

What is the purpose of the Gel Electrophoresis lab?

🎯 Purpose of Gel Electrophoresis: To analyze DNA size (e.g., restriction digest fragments, PCR products) To compare genetic material To verify successful DNA cloning or transformation

How do you keep from spreading COVID-19 illness to others if you are sick?

🏠 1. Stay Home Do not go to work, school, or public places. Stay home except to get medical care. Avoid public transportation, ride-sharing, and taxis. 😷 2. Isolate Yourself Stay in a separate room away from others in your household, if possible. Use a separate bathroom, if available. Avoid sharing personal household items (dishes, towels, bedding). 😷 3. Wear a Mask If you must be around others, wear a well-fitted mask. Make sure your nose and mouth are covered completely. Others in your home should wear masks too, especially in shared spaces. 💧 4. Cover Coughs and Sneezes Use a tissue or your elbow, not your hands. Throw away used tissues and wash your hands immediately.

How does absorbance change due to concentration in the graph when using a spectrophotometer graph?

📈 Directly proportional More bacteria → more light is scattered/absorbed → higher OD reading Fewer bacteria → less light is scattered/absorbed → lower OD reading

What do the results of the Kirby-Bauer tell us?

📏 What It Tells You: The larger the zone of inhibition = the more sensitive the bacteria are to that antibiotic and the stronger the effectiveness of the antibiotic. The smaller or absent zone = the bacteria may be resistant. This is the principle behind the Kirby-Bauer antibiotic sensitivity test.

How does it work?

🔁 How it works: Ampicillin present?→ Only bacteria with bla gene survive. Arabinose present?→ AraC protein activates the GFP gene, and cells glow green. _____________________________ +pGLO, +ampicillin = bacteria survive +pGLO, +ampicillin + arabinose = Bacteria survive and glow -pGLO (no plasmid) Bacteria die on amp plates = no glow

What are some uses for artificial gene transfer?

🔬 1. Genetic Engineering of Organisms GMOs (Genetically Modified Organisms): Example: Corn or soybeans engineered to resist pests or tolerate herbicides. Purpose: Increase crop yield, reduce pesticide use. 💉 2. Medicine and Gene Therapy Gene Therapy: Introducing correct genes into human cells to treat genetic disorders. Example: Treating cystic fibrosis or certain cancers. Insulin Production: Bacteria like E. coli are genetically modified to produce human insulin for diabetic patients. 3. Research Studying gene function: Scientists insert genes into model organisms (like mice or yeast) to study diseases or gene behavior. Reporter genes (like GFP - green fluorescent protein): Help visualize gene expression under a microscope. 4. Vaccine Development Example: Recombinant vaccines like Hepatitis B, where a viral gene is inserted into yeast cells to produce a harmless viral protein for immunization. 5. Industrial Microbiology Microbes engineered to produce: Enzymes (used in detergents, food processing) Biofuels (like ethanol) Bioplastics (eco-friendly plastics)

What are the advantages and disadvantages for each of the different ways to enumerate bacteria?

🔬 1. Standard Plate Count (SPC) Advantages: Counts only viable (living) cells that can reproduce Inexpensive Can be used to isolate pure colonies 🔴 Disadvantages: Takes 24+ hours for colonies to grow Cannot detect anaerobes well Cells that are clumped form one colony → underestimates total count Needs proper dilution for accurate results 🌫 2. Turbidity (Optical Density via Spectrophotometer) Advantages: Fast and easy Non-destructive (sample can be reused) Good for monitoring bacterial growth over time 🔴 Disadvantages: Indirect (doesn't differentiate live vs dead cells) Less accurate at high densities (light scattering errors) Requires a standard curve to estimate actual CFU/mL 🔬 3. Direct Microscopic Count Advantages: Fast Can see cell shape and arrangement Works for cells that can't grow on agar 🔴 Disadvantages: Counts both live and dead cells Small sample size = not always representative Difficult to count moving or clumped bacteria Needs specialized equipment (hemocytometer or Petroff-Hausser chamber) 💧 4. Filtration Method Advantages: Useful for very dilute samples (like water) Concentrates bacteria from large volumes Viable count (bacteria grow on the filter) 🔴 Disadvantages: Only works if bacteria can grow on selected medium Not ideal for samples with high bacterial load Can clog with particulate matter 📊 5. Most Probable Number (MPN) Advantages: Useful for samples where bacteria can't be plated (e.g., water, soil) Good for low numbers of organisms 🔴 Disadvantages: Less precise, gives a statistical estimate Time-consuming More steps (serial dilutions + multiple tubes)

What are the 3 important pathogens found in nose or throat and how to recognize on sheep blood agar:

🔬 1. Streptococcus pyogenes (Group A Strep) Hemolysis: Beta-hemolysis → clear, complete zone around colonies Colony Appearance: Small, translucent, sometimes dry-looking Importance: Causes strep throat, scarlet fever, rheumatic fever Clue: The strong clear zone is the key sign on SBA 🔬 2. Staphylococcus aureus Hemolysis: Often beta-hemolytic, but not always Colony Appearance: Large, round, golden-yellow colonies Importance: Can cause skin infections, pneumonia, toxic shock Clue: Yellowish pigment + beta-hemolysis suggests S. aureus (Confirm with coagulase test if needed) 🔬 3. Streptococcus pneumoniae Hemolysis: Alpha-hemolysis → greenish discoloration around colonies Colony Appearance: Small, dome-shaped early, may flatten with age Importance: Causes pneumonia, sinusitis, ear infections, meningitis Clue: Look for greenish halo, and sometimes a dimpled center of colonies