MBB 110.1 Lab Cheatsheet

Cheatsheet Content

### Bacterial Genotypes Review - **Key Concepts:** Strain definition, common *E. coli* lab strains (MG1655, DH5α, S17-1 λpir, BL21(DE3)), their specific mutations, and why these mutations are significant for their applications (e.g., cloning, protein expression, conjugation, blue-white screening). - **MG1655:** Wildtype-like, mild autotroph. What does *ilvG-*, *rfb-50*, *rph-1* mean? - **DH5α:** Molecular cloning. Explain *Δ(lacZYA-argF)U169*, *φ80dlacZΔM15*, *purB20*. How do these relate to blue-white screening and heterotrophy? - **S17-1 λpir:** Bacterial conjugation. What is *RP4-2*? Why *recA1 endA1 hsdR17*? - **BL21(DE3):** Protein overexpression. Explain *lon-11 Δ(ompT-nfrA)885*, *hsdS10*, and the role of *λDE3* (T7 RNA polymerase). - **Application Questions:** 1. Why is DH5α a preferred strain for cloning experiments involving blue-white screening, and what would happen if you used MG1655 instead? 2. You want to overexpress a recombinant protein in *E. coli* and maximize its yield by preventing degradation. Which *E. coli* strain would you choose and why, based on its genetic characteristics? 3. A research group is attempting to transfer a plasmid from one bacterial strain to another via conjugation. They choose S17-1 λpir as the donor. What specific genetic features of S17-1 λpir make it suitable for this purpose? 4. If a strain is described as *purB20*, what does this imply about its nutritional requirements, and how would you prepare a minimal media to grow it? ### Culture Media Review - **Key Concepts:** Types of media (solid/liquid), components of common rich media (LB, Nutrient Broth, TSB) and their functions (Tryptone, Yeast Extract, NaCl, Peptone, Beef Extract, Soytone). Agar properties (hysteresis, inertness). - **LB Formulations:** Lennox, Miller, Luria – what's the difference and when would you use each? - **Agar:** What is it, why is it used, and what does "hysteresis" mean in this context? - **Application Questions:** 1. You are preparing an LB medium for a bacterial culture that is known to be slightly halophilic. Which LB formulation would you choose and why? 2. A new researcher accidentally omits yeast extract from their LB medium preparation. What impact would this likely have on bacterial growth, and why? 3. Explain the importance of agar in solid media. If agar did not exhibit hysteresis, how might it complicate laboratory procedures? 4. You are tasked with growing a fastidious pathogenic bacterium. Which type of rich medium (LB, Nutrient Broth, TSB) would be most suitable, and what specific components in that medium cater to its needs? ### Bacterial Isolation & Preservation Review - **Key Concepts:** Colony formation, streak/spread plate methods, pure culture definition and verification. Limitations of isolation (fastidious, slow-growing, extremophiles, oxygen). Preservation methods (paraffin, lyophilization, cryopreservation) and their principles (vitrification, cryoprotectants). Storage temperatures and their implications. - **Application Questions:** 1. You perform a streak plate and observe colonies that appear different in color and morphology. What is your next step to ensure you have a pure culture of your desired bacterium, and why is this step important? 2. A novel bacterium is discovered that grows optimally at 90°C and requires a specific, complex mixture of deep-sea minerals. Discuss the challenges you would face in isolating and culturing this bacterium in a laboratory setting. 3. You need to store a bacterial strain for several years without losing viability. Which preservation method would be most appropriate, and what is the critical component (and its function) required for this method to be successful? 4. Explain why storing bacterial cultures at -20°C without cryoprotectants is generally not recommended for long-term viability, even though it's a "freezer." ### Oxygen Utilization Review - **Key Concepts:** Purpose of oxygen utilization tests (e.g., thioglycolate medium). Classifications of bacteria based on oxygen requirements (obligate aerobe, facultative anaerobe, obligate anaerobe, microaerophile, aerotolerant anaerobe). ROS adaptation and scavenging mechanisms (SOD, catalase, GPX, PRDX, antioxidants). - **Application Questions:** 1. You observe growth only at the very top surface of a thioglycolate medium. What is the oxygen requirement of this bacterium? If this bacterium is also known to produce catalase, how does this enzyme contribute to its survival? 2. A bacterium is classified as a facultative anaerobe. Describe where you would expect to see its growth in a thioglycolate tube, and explain its metabolic flexibility regarding oxygen. 3. Explain the detrimental effects of Reactive Oxygen Species (ROS) on bacterial cells. Outline at least two enzymatic mechanisms bacteria employ to detoxify ROS, including the enzymes and the reactions they catalyze. 4. *Vibrio campbellii* is a facultative anaerobe but is known to be sensitive to cold temperatures. How might its ROS adaptation mechanisms be affected at lower temperatures, and what implications does this have for its survival? ### Selective & Differential Media Review - **Key Concepts:** Definitions and examples of selective, differential, and enriched media. Specific components and their roles in MSA, MacConkey, EMB, Blood Agar, and TCBS. Understanding how each medium selects for certain bacteria and differentiates others. Minimal media components and use. - **Application Questions:** 1. You are given a mixed sample of Gram-positive *Staphylococcus aureus* (mannitol fermenter, halotolerant) and Gram-negative *E. coli* (lactose fermenter). Which two different agar types would you use to isolate and differentiate these two species, and what would be the expected appearance of each bacterium on these plates? 2. A patient presents with symptoms suggestive of a *Vibrio* infection. Which specialized agar would a clinical microbiologist use to isolate and identify the pathogen, and what specific features of this agar allow for both selection and differentiation of *Vibrio* species? 3. Explain the dual roles of eosin Y and methylene blue in EMB agar. How do these components contribute to both the selective and differential properties of the medium? 4. You are attempting to isolate an autotrophic bacterium from a soil sample. What type of media would you prepare, and what are the essential components you would include, ensuring it only provides the "bare necessities"? ### Antibiotic Selectivity Review - **Key Concepts:** Mechanisms of action for beta-lactam antibiotics, aminoglycosides, chloramphenicol, and tetracycline. Understanding the bacterial ribosome structure (50S, 30S, A/P/E sites). Bacterial resistance mechanisms (chemical neutralization, efflux pumps, alteration of binding site) and specific resistance genes (*bla*, *aphA*, *tetA*, *cat*, *aadA1*). - **Application Questions:** 1. A newly discovered antibiotic is found to bind to the 30S ribosomal subunit, preventing tRNA from entering the A site. Classify this antibiotic by mechanism of action, and predict whether it would be bacteriostatic or bactericidal. 2. You are working with an *E. coli* strain that is resistant to ampicillin. Describe the most common genetic mechanism for this resistance, including the gene responsible and its biochemical action. 3. Explain how tetracycline and chloramphenicol, both protein synthesis inhibitors, differ in their specific binding sites and effects on the ribosome. 4. If a bacterium acquires a plasmid containing the *aphA* gene, what antibiotic would it become resistant to, and what is the enzymatic function of the product of this gene? ### Blue-White Screening Review - **Key Concepts:** Purpose of blue-white screening in cloning. Roles of IPTG and X-gal. Mechanism of *lacZ* complementation in DH5α. Interpreting results (blue vs. white colonies) in the context of successful cloning and plasmid insertion. - **Application Questions:** 1. You perform a blue-white screen after ligating a foreign DNA fragment into a plasmid. You observe both blue and white colonies on your plate. Which colonies would you pick for further analysis, and what does their color indicate about the success of your ligation? 2. Explain the specific role of IPTG in the blue-white screening process. What would happen if you forgot to add IPTG to your agar plates? 3. A student performs blue-white screening but accidentally uses a non-competent *E. coli* strain that lacks the *lacZΔM15* mutation. What would be the expected outcome on the selection plates, assuming transformation still occurs? 4. You are given a plate from a blue-white screening experiment designed to identify recombinant plasmids. You notice that all colonies are blue, and there are very few colonies overall. What two common issues in the cloning process could lead to this result? ### Staining & Motility Review - **Key Concepts:** Purpose of staining (visualization, specific properties). Components of staining (primary, secondary, mordant, fixer, decolorizer). Gram staining procedure and interpretation. Capsule staining (Anthony's, Maneval's) and their principles (negative staining, contrast). Bacterial motility (planktonic, chemotaxis, swimming, swarming) and factors affecting it (agar concentration, shape). - **Application Questions:** 1. Describe the step-by-step procedure for Gram staining. For each step, explain the reagent used and its specific function in differentiating Gram-positive and Gram-negative bacteria. 2. You observe a bacterial cell under a microscope after performing Anthony's capsule stain, and it appears as a clear halo around a purple cell against a dark background. Explain the principle behind this observation, and why heat-fixing is avoided in capsule staining. 3. A microbiologist is trying to determine if a newly isolated bacterium is motile. They inoculate the bacterium into a semi-solid agar medium (0.3% agar). If the bacterium is motile, what pattern of growth would be observed, and how does this differ from growth in a solid agar medium (1.5% agar)? 4. Compare and contrast swimming and swarming motility in bacteria. What environmental factor commonly differentiates these two types of movement in a laboratory setting? ### Bacterial Growth Curve Review - **Key Concepts:** Phases of bacterial growth (lag, exponential, stationary, death) and the characteristics of each. OD600 measurement (principle, limitations, comparison with CFU/mL). Abiotic factors affecting growth (temperature, osmolarity, pH) and mechanisms of adaptation. Biotic factors (species, contamination, protein expression). - **Application Questions:** 1. You are monitoring bacterial growth using OD600 readings. After 8 hours, your readings consistently show values above 1.0. What is the appropriate action to take, and why is it necessary? 2. A bacterium is described as a psychrophile. What does this mean for its optimal growth temperature, and what cellular adaptations would it likely possess to thrive in such conditions? 3. Explain why a bacterial culture might enter the stationary phase even if fresh nutrients are continuously supplied, assuming it's a batch culture without an outlet. What are the primary limiting factors in this scenario? 4. You are investigating a bacterial population in a continuous culture system (chemostat) where nutrients are constantly supplied, and waste products are removed. However, you notice a sudden drop in bacterial density. Propose two distinct biotic factors that could explain this decline.