Lab Flashcards

Question

PEA Observations Conclusion

Answer 1

- *Type*: **Selective and Differential Medium** **Selective Function** - *Selective agents*: **Bile salts** and **crystal violet** - *Mechanism*: - Inhibit **Gram-positive** bacteria by disrupting peptidoglycan synthesis - **Gram-negative** bacteria are protected by their outer membrane → grow well **Differential Function** - *Differential agents*: **Lactose** (fermentable sugar) and **neutral red** (pH indicator) - *Mechanism*: - Fermentation of lactose produces **acid**, lowering pH - **Neutral red** turns **colonies pink** at pH < 6.8 - No fermentation → no acid → colonies remain **colorless**

Answer 2

*Step 1: Evaluate Selective Growth* - **Good growth** → Organism is **Gram-negative** - **Poor or no growth** → Organism is **Gram-positive** (inhibited) *Step 2: Evaluate Differential Results (if growth occurred)* - **Pink colonies** → Organism **ferments lactose** (acidic pH) - **Colorless colonies** → Organism **does not ferment lactose** *Important*: - If no growth occurs → no differential interpretation can be made.

Answer 3

- *Type*: **Selective and Differential Medium** **Selective Function** - *Selective agent*: **7.5% NaCl** - *Mechanism*: - Selects for **halotolerant** organisms (can withstand high salt) - Inhibits **non-halotolerant** organisms → poor or no growth **Differential Function** - *Differential agents*: **Mannitol** (fermentable sugar) and **Phenol Red** (pH indicator) - *Mechanism*: - Fermentation of mannitol → acid production → **lowers pH** - **Phenol Red** color shifts: - **< 6.8** → Yellow (acidic) - **6.8–8.2** → Red (neutral) - **> 8.2** → Pink (basic) - *Result*: Yellow medium = mannitol fermentation occurred

Answer 4

1. **Poor or no growth** → **Conclusion**: Organism is **not halotolerant** 2. **Good growth, medium remains pink/red** → **Conclusion**: Organism is **halotolerant**, but **does not ferment mannitol** 3. **Good growth, medium turns yellow** → **Conclusion**: Organism is **halotolerant** and **ferments mannitol**

Answer 5

- *Type*: **Differential medium** - *Purpose*: Distinguishes organisms based on **oxygen requirements** (e.g., obligate aerobe vs. facultative anaerobe) **Key Components and Functions** 1. **Sodium thioglycollate & L-cystine** - *Function*: **Oxygen scavengers** → bind free O₂ to create anaerobic conditions 2. **Agar (small amount)** - *Function*: Increases viscosity → prevents mixing → preserves distinct **aerobic (top)** and **anaerobic (bottom)** zones 3. **Resazurin** (oxygen indicator) - *Function*: Indicates O₂ presence by **color change** - **Purple** = oxygen present (aerobic) - **Colorless** = no oxygen (anaerobic) **Mechanism Summary** - Medium is **fully anaerobic** after autoclaving - Upon cooling, **oxygen diffuses from top downward** - Creates a **gradient**: - **Top = aerobic** (purple) - **Bottom = anaerobic** (colorless) - Growth location in tube → indicates organism's oxygen requirement

Answer 6

- *Observation*: **Location of cloudiness** (turbidity) in the tube indicates oxygen requirements. 1. **Cloudiness only in aerobic (top) zone** → **Conclusion**: Organism is an **obligate aerobe** → Does **not** grow anaerobically 2. **Cloudiness in both aerobic and anaerobic zones** → **Conclusion**: Organism is a **facultative anaerobe** → Can grow **with or without oxygen**

Answer 7

- *Type*: **Differential test** - *Purpose*: Detects if an organism produces the **catalase enzyme**, which breaks down harmful **peroxide anions (H₂O₂)** into **water and oxygen gas** **Mechanism** - Add **hydrogen peroxide** to bacterial culture - If catalase is present → **oxygen bubbles** form - If catalase is absent → **no bubbles** **Interpretation** - **Bubbles** → Organism produces catalase - **No bubbles** → Organism does not produce catalase **Note**: - Catalase presence shows the organism can detoxify peroxides - It does **not** indicate oxygen usage type (aerobic vs. anaerobic)

Answer 8

- *Type*: **Differential test** - *Purpose*: Helps distinguish between **Gram-positive** and **Gram-negative** bacteria based on **cell wall lysis** **Mechanism** - Mix bacterial cells with **potassium hydroxide (KOH)** - **Gram-negative** cell walls lyse → DNA released → **stringy/viscous** solution - **Gram-positive** cell walls remain intact → **watery** solution **Interpretation** - **Viscous/gooey KOH** → **Gram-negative** (DNA released) - **Thin/watery KOH** → **Gram-positive**

Answer 9

- *Definition*: Determining the number or concentration of bacterial cells in a sample. - *Purpose*: Used to monitor microbial load for health, safety, and industry compliance (e.g., food, cosmetics, water)

Answer 10

dilute bacterial concentration in a stepwise manner for accurate colony counting. When performing the serial dilution you much use a fresh pipette tip in between each serial transfer. Make sure to gently invert each test tube before taking sample. When plating TSA plates keep plates LID SIDE UP. If you start by platting the most dilute sample first you do not need a new pipette. **Individual Dilution Factor** - *Formula*: Volume added ÷ total volume in tube - *Example*: 1 mL into 9 mL → 1 / (1+9) = 1/10 **Final (Overall) Dilution Factor** - *Formula*: Product of all individual dilution factors - *Example*: Tube 3 in 1:10 series → 1/10 × 1/10 × 1/10 = 1/1000

Answer 11

concentration of cells = (number of colonies on plate) / (final dilution factor of tube * volume plated mL) *valid CFU between 30-300*

Answer 12

- *Definition*: Enzymes produced inside the cell and secreted outside to act in the environment. - *Function*: Break down macromolecules into monomers for transport or act as virulence factors. **Examples of exoenzymes**: 1. **Amylase** and **glucosidase** - Hydrolyze amylose (starch) → maltose + glucose - Used to identify bacteria that can digest starch 2. **Collagenase** (from *Clostridium perfringens*) - Breaks down collagen in connective tissue 5. **Gelatinase** - Hydrolyzes gelatin (denatured collagen) into amino acids - Part of the **protease** family (enzymes that degrade proteins)

Answer 13

Fermentation is a metabolic process which allows an organism to oxidize NADH to NAD+ using an organic molecule as the final electron acceptor.

Answer 14

- **Type**: Differential medium - **Purpose**: Detect exoenzyme activity (amylase, glucosidase) → starch hydrolysis - **Process**: 1. **Soluble starch** = differential component 2. Bacteria secrete **amylase**/**glucosidase** → digest starch 3. Add **Gram’s iodine** → starch turns **blue-black** - Zone around starch-hydrolyzing growth appears **clear** - **Zone of clearing** = indicates **starch hydrolysis** and presence of exoenzymes

Answer 15

**Starch Agar: Observing and Interpreting Results** - **Observation**: Look for a **zone of clearing** around bacterial growth - Compare area near growth to area with no growth after iodine is added - **Interpretation**: - **Zone of clearing** present → organism hydrolyzes starch (produces **amylase**/**glucosidase**) - **No clearing** → organism does not hydrolyze starch (no enzyme secretion)

Answer 16

- *Type*: Complex, differential medium - *Purpose*: Detect ability to hydrolyze **gelatin** using exoenzyme **gelatinase** - *Gelatin origin*: Derived from collagen (animal connective tissue) - *Mechanism*: - If organism produces **gelatinase**, it hydrolyzes gelatin into polypeptides → liquid medium at room temp - No gelatinase → gelatin remains intact → medium stays solid - **Important**: Must observe **after cooling** to room temp (~25–28°C) or false positives may occur

Answer 17

**Nutrient Gelatin – Results** - *Observation*: Check consistency of the medium after incubation and cooling - *Interpretation*: - If **medium is solid** → organism does **not** hydrolyze gelatin OR does **not** produce gelatinase - If **medium is liquid** → organism **hydrolyzes gelatin** OR **produces gelatinase**

Answer 18

- *Purpose*: Distinguishes between organisms based on **fermentation pattern** - *pH indicator*: **Phenol red** - Yellow at pH < 6.8 → **acid** produced - Red/pink at higher pH → **no acid** - *Carbohydrate fermentation* produces: - **Acid** → phenol red turns yellow - **Gas** → trapped in inverted **Durham tube**, visible bubble forms - If incubated too long: - Organism depletes carbs, metabolizes peptones → releases **ammonia** - pH rises at surface → **red layer** on top of yellow broth - Acid fermentation still occurred

Answer 19

**Phenol Red Carbohydrate Broth – *Observations & Interpretation*** - *Observe*: - Color of broth → **Yellow = acid fermentation** - Bubble in Durham tube → **Gas production** - *Conclude*: - **Red/orange/pink broth, no bubble** → No fermentation of sugar - **Yellow broth, no bubble** → Acid fermentation only - **Yellow broth, bubble present** → Acid and gas fermentation - *Always include name of sugar tested* (e.g., glucose, lactose, sucrose)

Answer 20

- *Purpose*: Differential medium used to detect 1. **Sulfide** production (H₂S) 2. **Indole** production 3. **Motility** - *Sulfide Production* → Sodium thiosulfate reduced by some organisms to **H₂S** → H₂S reacts with **ferrous ammonium sulfate** → forms **black precipitate** - *Indole Production* → Requires tryptophanase to break tryptophan into **indole** → Add **Kovac's reagent** after incubation → Pink layer = **indole positive** - *Motility* → 3.5% agar limits diffusion → **Motile** organisms grow beyond stab line → **Non-motile** organisms grow only in stab → **Obligate aerobes** may yield false negatives (don’t grow well in stab)

Answer 21

**SIM Medium – Observations & Interpretation** - *Motility* - Growth/cloudiness **beyond stab** → **Motile** - Growth **confined to stab** → **Non-motile** - *Sulfide* - **Black precipitate** present → **Sulfide produced** - No precipitate → **No sulfide** - *Indole* (add 5 drops Kovac’s reagent) - **Pink top layer** → **Indole produced** - **Yellow/no change** → **No indol

Answer 22

- *Types*: 1. **Antibiotics** = *naturally occurring antibacterials* - Produced by bacteria (e.g. *Streptomyces*) and fungi (e.g. *Penicillium chrysogenum*) - Includes streptomycin, tetracycline, neomycin - Some also have antifungal (e.g. amphotericin B), antiparasitic (e.g. ivermectin), or antitumor effects 2. **Semisynthetic antibacterials** - Made by **modifying natural antibiotics** in the lab (e.g. ampicillin) - Improves spectrum, overcomes resistance 3. **Synthetic drugs** - **Fully engineered in lab**, not derived from natural products - Designed to target specific microbial processes

Answer 23

1. *Selective Toxicity* - Drug harms **microbe**, not host - Achieved by targeting structures **not found in humans** (e.g. peptidoglycan) - Example: **Penicillin** blocks peptidoglycan synthesis → no effect on humans 2. *Mode of Action* - Mechanism by which drug **affects the target cell** - **Antibacterials**: - Inhibit **protein synthesis** (70S ribosomes) - Inhibit **peptidoglycan synthesis** - Disrupt **cell membranes (LPS)** - **Antifungals**: Disrupt **ergosterol** or **glucan** synthesis - **Antivirals**: Inhibit **nucleic acid synthesis**, block **release** (e.g. Tamiflu) 3. *Spectrum of Activity* - **Narrow-spectrum**: Few organisms (e.g. **penicillin** → Gram+ only, **isoniazid** → *Mycobacterium*) - **Broad-spectrum**: Many organisms (e.g. **ciprofloxacin** → Gram+ and Gram−) - Broad-spectrum often used when **pathogen is unknown**

Answer 24

- Broad class of antibiotics that **interfere with bacterial cell wall synthesis** - Act by binding to **penicillin-binding proteins (PBPs)** → PBPs are bacterial enzymes that form **cross-links in peptidoglycan** → Blocking PBPs prevents cell wall synthesis → **cell lysis and death**, especially in actively dividing bacteria - Includes two main subclasses: **Penicillins** and **Cephalosporins** 1. **Penicillin (P)** - Classic beta-lactam - Effective mainly against **Gram-positive bacteria** 2. **Amoxicillin with Clavulanic Acid (AmC)** - **Amoxicillin** is a penicillin derivative with broader spectrum - **Clavulanic acid** inhibits **beta-lactamases** (enzymes that destroy beta-lactam antibiotics) - Used together to extend effectiveness against **beta-lactamase–producing bacteria** 3. **Ceftriaxone (CRO)** - A **cephalosporin**, another class of beta-lactam - Broad-spectrum: active against **Gram-positive and Gram-negative bacteria** - Often used for **serious infections** (e.g. meningitis, gonorrhea)

Answer 25

- Class of **polypeptide antibiotics** that **interfere with bacterial cell wall synthesis** - Act by **blocking transport of peptidoglycan precursors** across the cytoplasmic membrane → Prevents building blocks from reaching the cell wall → **Inhibits synthesis of new peptidoglycan**, weakening the wall and leading to cell death - Effective mainly against **Gram-positive bacteria** - Commonly used **topically** (e.g. in Neosporin®, Polysporin®) 1. **Bacitracin (B)** - Produced by *Bacillus subtilis* - Used to treat **skin infections** - Often combined with **neomycin** and/or **polymyxin B** in topical ointments

Answer 26

**Tetracyclines** - Broad-spectrum antibiotics that **inhibit bacterial protein synthesis** - Bind to the **30S ribosomal subunit** → Prevent attachment of **aminoacyl-tRNA** to the **A site** → Blocks elongation step of translation → **halts protein production** - Effective against a wide range of **Gram-positive and Gram-negative** bacteria 1. **Tetracycline (Te)** - Naturally occurring antibiotic from *Streptomyces* species - Used to treat infections like **acne**, **chlamydia**, **Rickettsial diseases** 2. **Doxycycline (D)** - **Semisynthetic** derivative of tetracycline - Longer half-life, better absorption - Commonly used for **Lyme disease**, **malaria prophylaxis**, **acne**, and **respiratory infections**

Answer 27

- Class of antibiotics that **inhibit protein synthesis** - Bind to the **50S ribosomal subunit** → This blocks **peptide bond formation** during elongation of the growing polypeptide → Result: **prevents bacterial protein synthesis** and stops bacterial growth - Derived from natural product **lincomycin**, modified to increase effectiveness 1. **Clindamycin (CC)** - **Semi-synthetic derivative** of lincomycin - Produced by *Streptomyces lincolnensis* - Broad-spectrum against **anaerobic bacteria** and some **Gram-positive cocci**

Answer 28

- Class of **synthetic antibacterials** that inhibit **DNA replication** - Bind to **DNA gyrase**, a bacterial topoisomerase → DNA gyrase relieves supercoiling during replication → Inhibition traps the enzyme on DNA, leading to **topoisomerase poisoning** → Blocks DNA replication and transcription → **cell death** - Effective against a broad range of **Gram-positive and Gram-negative bacteria** 1. **Ciprofloxacin (CIP)** - Commonly used fluoroquinolone - Broad-spectrum activity - Often used for **urinary tract infections**, **respiratory infections** 2. **Levofloxacin** - Another fluoroquinolone - Similar mechanism and spectrum to ciprofloxacin

Answer 29

- **Synthetic antibiotics** that interfere with **folic acid biosynthesis** in bacteria - Act as **competitive inhibitors** of **dihydropteroate synthetase** → Prevent conversion of PABA to **dihydropteroic acid**, an early folate precursor → Humans don’t synthesize folic acid, so this pathway is unique to bacteria → selective toxicity - Inhibiting folate synthesis disrupts DNA replication and transcription* - Commonly combined with **trimethoprim** to increase effectiveness and reduce resistance - **Sulfamethoxazole** + **Trimethoprim** = **SxT (Bactrim)**

Answer 30

- **Synthetic antibiotic** that targets **folate biosynthesis** in bacteria - Acts as a **competitive inhibitor** of **dihydrofolate reductase** → Prevents conversion of **dihydrofolic acid** to **tetrahydrofolic acid** → This blocks nucleotide synthesis → disrupts **DNA replication and cell division** - Used in **combination therapy** with sulfonamides (e.g. sulfamethoxazole) → Each drug targets a different enzyme in the same pathway → Synergistic effect reduces resistance and improves efficacy - Part of **SxT (Bactrim)** combo

Answer 31

- Used to test **antimicrobial susceptibility** of a bacterial strain - **Paper disks** containing antimicrobial drugs are placed on a freshly swabbed **Mueller-Hinton agar** plate - The antimicrobial **diffuses outward**, forming a **concentration gradient** - If the bacterium is **susceptible**, a **zone of inhibition** appears around the disk → Indicates growth has been inhibited during incubation

Answer 32

- Standard **growth medium** used for Kirby-Bauer assay - Supports consistent diffusion of antibiotics - **Ingredients (per liter)**: - 17.5 g acid hydrolysate of casein - 2 g beef extract - 1.5 g starch - 17 g agar

Answer 33

- Observe for **presence or absence** of a **zone of inhibition** - Use **(–)** symbol if no zone is observed - **Measure the diameter** of any zone present (in **millimeters**) - Larger zones = greater effectiveness *Conclusions*: - **No zone of inhibition** → organism is **resistant** - **Larger zone** → organism is **more susceptible** - Testing against **multiple organisms** helps determine **spectrum of activity**

Lab Flashcards

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