Unit 3 - Practice Quiz

Bacteria are single-celled organisms that lack a nucleus and other membrane-bound organelles, which is the defining characteristic of prokaryotes. Fungi, protozoa, and algae are all eukaryotes.

Viruses are infectious agents made of a core of nucleic acid (either DNA or RNA) surrounded by a protective protein shell called a capsid. They are not cells and require a host to replicate.

Serial dilution is a technique used to systematically decrease the concentration of a dense culture of microbes, making it possible to get a countable number of colonies on an agar plate.

The dilution factor is the volume of the sample divided by the total volume (sample + diluent). Here, it is 1 mL / (1 mL + 9 mL) = 1/10, which is a 1:10 dilution.

In the pour plate method, a known volume of the diluted sample is pipetted into an empty Petri dish, and then molten, cooled agar is poured over it and mixed.

Because the sample is mixed directly with the molten agar, microorganisms are distributed throughout the medium, allowing colonies to develop both on the surface and within the agar.

The streak plate method is a qualitative isolation technique used to separate individual microbial cells on an agar plate, so they can grow into distinct, isolated colonies.

An inoculating loop, a tool with a small wire loop at the end, is used to pick up the inoculum and spread it across the agar surface in a specific pattern to achieve isolation.

In the spread plate method, a liquid sample is spread over the surface of an already solidified agar plate. Consequently, all resulting colonies grow exclusively on the agar surface.

A sterile, L-shaped tool, commonly called a cell spreader or hockey stick, is used to evenly distribute the liquid inoculum across the entire surface of the agar plate.

Nutrient broth is a liquid growth medium. Nutrient agar is the solid equivalent, containing agar as a solidifying agent.

Agar is a polysaccharide derived from seaweed that is used to solidify liquid media. Most microorganisms cannot metabolize it, so it provides a stable, solid surface for growth.

Plate count methods only count viable (living) cells that are capable of growing and forming a visible colony. The result is often expressed as Colony Forming Units (CFU) per mL.

A direct microscopic count involves placing a liquid sample on a hemocytometer (a slide with a grid of known dimensions) and counting the cells under a microscope. This method counts both live and dead cells.

The log (or exponential) phase is the period of most rapid growth, where bacteria are actively dividing and the population size doubles at a regular interval.

The stationary phase is reached when nutrient depletion and waste accumulation cause the growth rate to slow down, resulting in a plateau where the number of new cells produced balances the number of cells dying.

The primary cause of food spoilage is the growth and metabolic activity of microorganisms such as bacteria, yeasts, and molds, which alter the food's texture, taste, smell, and appearance.

Visible signs like the growth of fuzzy mold, the development of a slimy texture, or the production of off-odors and gas are classic indicators of spoilage caused by microorganisms.

Low temperatures in a refrigerator do not kill most microbes but instead slow down their metabolic processes and rate of reproduction, thereby extending the time it takes for food to spoil.

Canning involves heating food to high temperatures in a sealed container. This process kills spoilage-causing microorganisms and their spores, and also inactivates enzymes that can degrade the food.

Each transfer is a 1:10 or dilution. Four such dilutions result in a total dilution factor of . The original concentration is calculated as: (Number of colonies) / (Volume plated in mL × Total dilution factor) = CFU/mL.

Streak plating is the primary method used to separate microorganisms from a mixed culture to obtain pure, isolated colonies. The technique mechanically dilutes the inoculum over the agar surface, allowing individual cells to form distinct colonies.

Primary metabolites are produced during the exponential (log) growth phase. However, secondary metabolites like many antibiotics are often produced when growth slows down due to nutrient limitation or waste accumulation, which occurs during the stationary phase.

Pasteurization uses moderate heat to eliminate specific pathogens and reduce spoilage organisms. Unlike sterilization, which kills all microbes but can significantly alter the taste, color, and nutritional content, pasteurization preserves the sensory qualities of the food.

The classification is based on its environmental tolerances and metabolism: 'Thermo-' for high temperature, 'acido-' for low pH, 'chemo-' for using chemical energy, and 'autotroph' for using an inorganic carbon source (CO₂).

In pour plating, the inoculum is mixed with molten agar. This entraps bacteria within the agar medium where oxygen levels are very low, creating an anaerobic environment suitable for the growth of obligate anaerobes, in addition to facultative anaerobes on and near the surface.

Turbidity measurement is an indirect method that is very rapid. It measures the amount of light scattered by the cells (optical density), which correlates to cell concentration. The other methods are either direct counts or require a lengthy incubation period.

Semi-solid agar has a lower concentration of agar, creating a soft gel. Motile bacteria can move through this medium, away from the initial stab inoculation line, creating a visible turbidity or cloudiness that indicates motility. Standard agar is too firm for this movement.

The number of generations (n) is the total time divided by the generation time: n = 120 minutes / 20 minutes/generation = 6 generations. The final population is . So, or cells/mL.

HPP uses immense pressure to disrupt microbial cell membranes and enzymes, effectively pasteurizing the product. Because it is a non-thermal process, it avoids the heat-induced degradation of vitamins, pigments, and flavor molecules common in traditional pasteurization.

Direct microscopic counting visualizes all cells present, regardless of whether they are alive or dead. A standard plate count only enumerates viable cells capable of growing and forming a colony. In an old biofilm with many dead cells, this discrepancy is expected.

Two fundamental differences between Archaea and Bacteria are cell wall and membrane composition. Bacteria have peptidoglycan in their cell walls and ester-linked membrane lipids, whereas Archaea lack peptidoglycan and have unique ether-linked lipids, which provides stability in extreme environments.

Failure to change tips results in carryover of cells from a more concentrated tube to the next, more dilute tube. This makes the subsequent dilutions more concentrated than they are calculated to be. A higher cell concentration leads to more colonies on the final plate, which, when multiplied by the large dilution factor, results in an artificially high (overestimated) original concentration.

Psychrophilic organisms are adapted to cold temperatures and can be sensitive to heat. Even brief exposure to the ~45-50°C temperature of molten agar before it solidifies can be lethal to some of these cells, leading to an underestimation of their numbers in the sample.

A selective agent inhibits the growth of some organisms while allowing others to grow. Here, bile salts and crystal violet inhibit Gram-positive bacteria, thus 'selecting for' the growth of Gram-negative bacteria like coliforms. The lactose and pH indicator are differential agents, distinguishing between lactose fermenters and non-fermenters.

Exponential growth is defined by the equation , where N is the number of cells and is the specific growth rate. Since is constant during this phase, the overall growth rate () is directly proportional to the population size (N). A larger population grows faster in absolute numbers.

Lactic Acid Bacteria metabolize the milk sugar, lactose, producing lactic acid as a byproduct. The accumulation of lactic acid lowers the milk's pH, which causes the primary milk protein, casein, to denature and coagulate, resulting in the characteristic sour taste and thickened texture (curdling).

The goal of spreading is to evenly distribute the inoculum across the entire agar surface. If the plate is not turned or the spreader is not used effectively, the liquid sample will remain concentrated in one area, leading to dense, localized colony growth instead of well-isolated colonies.

For statistical accuracy in plate counts, a plate with 30 to 300 colonies is used. Plates with >300 colonies are too numerous to count (TNTC) and may have competition effects. Plates with <30 colonies are subject to large statistical errors. Therefore, the plate with 150 colonies is the ideal choice.

Sodium benzoate is the salt of benzoic acid. In an acidic environment (low pH), it exists primarily in its undissociated, more lipid-soluble form (benzoic acid). This form can readily penetrate the cell membrane of microbes. Once inside the more neutral cytoplasm, it dissociates and disrupts metabolic functions, inhibiting growth.

At steady state in a chemostat, the specific growth rate equals the dilution rate D. So, . Using the Monod equation, , we solve for the steady-state substrate concentration S: , which gives S = 0.333 g/L. The cell concentration X is then calculated using the yield coefficient: g/L.

A bacteriostatic antibiotic inhibits bacterial reproduction but does not kill the cells. Therefore, the total number of cells (both living and non-dividing) remains high, which is measured by the direct microscopic count. However, since the cells cannot form colonies, the viable plate count will be very low or zero. A bactericidal agent would cause both counts to drop.

Flat sour spoilage in low-acid canned foods is characteristic of thermophilic, spore-forming bacteria that ferment carbohydrates to produce acid without gas. Bacillus stearothermophilus (now Geobacillus stearothermophilus) is a classic example. Clostridium species typically produce gas, causing cans to swell. Lactobacillus is generally not thermophilic enough to survive canning, and yeast (Saccharomyces) would produce gas (CO2).

The characteristics described (extremophile, ether-linked lipids, no peptidoglycan) are hallmarks of the domain Archaea. While these phenotypic traits are strong indicators, the most definitive and universally accepted method for establishing phylogenetic relationships among prokaryotes is by comparing the nucleotide sequences of conserved molecules, with the 16S ribosomal RNA gene being the gold standard.

Diauxic growth occurs because bacteria utilize the preferred energy source (glucose) first. Glucose metabolism represses the genes for metabolizing other sugars like lactose via catabolite repression. The intermediate lag phase is the time required for the cell to synthesize the necessary enzymes (like β-galactosidase and permease from the lac operon) to metabolize lactose once the glucose is fully depleted.

Pour plating involves mixing the inoculum with molten agar at ~45-50°C, which could kill or injure heat-sensitive (psychrophilic or mesophilic) organisms. It also results in colonies embedded in the agar where oxygen is limited, which is unsuitable for an obligate aerobe. Spread plating keeps the cells on the surface of pre-poured, cooled agar, ensuring their viability and access to oxygen, making it the ideal choice.

The medium must be selective and support the growth of only the desired strain. It needs to be a defined minimal medium to select against prototrophs that might be present. It must contain ampicillin to kill non-resistant cells. Crucially, it must contain histidine because the target strain is an auxotroph and cannot grow without it. A medium lacking histidine would fail to grow the desired strain.

Spectrophotometry for bacterial cultures measures light scattering, not true absorbance. The Beer-Lambert law assumes that photons are either transmitted or absorbed/scattered by a single particle. At high cell densities, the probability of multiple scattering events increases. Light scattered away from the detector by one cell can be scattered back towards it by another, causing the measured OD to be lower than predicted, breaking the linear relationship between OD and cell concentration.

The core principle of hurdle technology is that multiple mild preservation factors (e.g., slightly lowered pH, reduced water activity, MAP, mild heat) create a hostile environment for microbes. Microbes might overcome a single hurdle, but they cannot overcome multiple simultaneous stresses on their homeostasis. This synergistic effect allows for effective preservation without the harshness of a single high-intensity method, thus better retaining the food's nutritional value, flavor, and texture.

The statistically valid plate for counting is the one with 30-300 colonies. The plate with 28 colonies is acceptable, while the 315 is slightly over but less reliable than the 28. The TNTC plate is unusable. Using the plate with 28 colonies from the dilution: CFU/mL = (Number of colonies) / (Volume plated in mL) (Dilution factor). The original suspension had 1g in a total of 10mL, so this is the initial dilution. The plated dilution is , so the total dilution factor from the original solid is . Calculation: CFU/g = (28 colonies / 0.1 mL) = CFU/g. Wait, let me re-calculate the dilution. Initial suspension is 1g in 9mL, total volume is 10mL. So 1g/10mL is the initial step. This is often considered the dilution. Plating from the tube means the total dilution is followed by five more 10-fold dilutions, so it's a dilution overall from the initial slurry. Total dilution factor = . Original sample was 1g in 10mL. So concentration in the tube = Original CFU/g (1g/10mL) . This is getting complicated. Let's use the standard approach. Original suspension (1g in 9mL) is the dilution. Plating from tube gives a total dilution factor of . CFU/g = (Colonies) (Total Dilution Factor) / (Volume plated in mL initial g/mL). Let's simplify: CFU/g = (Colonies / Volume Plated in mL) (Dilution Factor of tube) (Initial Dilution Volume/Initial mass). CFU/g = (28 / 0.1) (10 mL / 1g) = CFU/g. This doesn't match the options. Let's re-read the common convention. The 1g in 9mL IS the dilution. The tube labeled has undergone 6 ten-fold dilutions. The total dilution factor is . CFU/g = (Colonies) / (Volume plated in mL Dilution). CFU/g = 28 / (0.1 mL ) = CFU/g. This is the standard, correct way. Let's check the 315 count. CFU/g = 315 / (0.1 mL * ) = CFU/g. The plate with 28 is statistically more sound as 315 is outside the ideal range. Therefore, CFU/g is the best answer derived from the most reliable plate.

The relationship between the specific growth rate () and the generation time ( or ) is given by the formula . Given and using , the generation time is calculated as: hours.

The current polyphasic approach to prokaryotic taxonomy uses specific cutoffs. A 16S rRNA sequence identity of >97% suggests the organisms might belong to the same species, but it is not definitive. The gold standard for species delineation is DNA-DNA hybridization, where a value of >70% relatedness is required to classify organisms as the same species. Since the hybridization value is only 45% (well below 70%), but the 16S identity is high (typically >95% for same genus), they are classified as belonging to the same genus but representing distinct species.

The purpose of flaming the loop between quadrants is to drastically reduce the number of cells being carried over. By not flaming the loop between quadrants 1 and 2, the analyst effectively dragged the same large, initial inoculum into the second quadrant. This results in confluent growth in both areas. Consequently, the starting inoculum for quadrant 3 (dragged from quadrant 2) is still far too high, leading to dense growth there as well and likely preventing the formation of well-isolated colonies in the final quadrant.

High-pressure processing inactivates microbes primarily by disrupting cell membranes and denaturing proteins. However, it has little effect on the smaller molecules held together by covalent bonds, such as vitamins (like Vitamin C), pigments, and flavor compounds. Thermal pasteurization uses heat, which can readily break down these sensitive compounds, altering the taste, color, and nutritional value of the juice. HPP is generally not effective against spores, and it achieves pasteurization, not sterilization.

A fastidious organism is one that has complex or particular nutritional requirements. TSB is a complex medium containing digests of proteins (peptones) and other extracts, providing a rich mixture of amino acids, vitamins, and growth factors. M9 is a defined minimal medium containing only a basic carbon source and essential salts. The ability to grow in the complex medium but not the minimal medium indicates the isolate cannot synthesize all of its required organic compounds (like specific amino acids or vitamins) from basic precursors and requires them to be supplied pre-formed.

The MPN method uses a series of liquid enrichment broths that can help resuscitate stressed or injured cells, allowing them to grow and produce a positive result (gas/acid). The membrane filtration technique places cells directly onto a selective and differential solid medium. Stressed cells, particularly those from treated water, may be too weakened to survive the additional stress of the selective agents on the solid agar and form a colony. This often leads to MPN giving a higher, more inclusive count of viable cells compared to MF.

In a zero-order reaction, the rate is independent of the reactant concentration. In the Monod equation, if the substrate concentration S is much greater than the half-saturation constant (i.e., ), the term () in the denominator becomes approximately equal to S. The equation then simplifies to . In this state, the growth rate reaches its maximum and is no longer limited by the substrate concentration, making it effectively a zero-order reaction with respect to S.

A facultative anaerobe can grow with or without oxygen, but typically grows much better with it because aerobic respiration yields significantly more ATP than fermentation or anaerobic respiration. Colonies on the surface have full access to atmospheric oxygen and can grow rapidly into large colonies. Colonies embedded within the agar are in an anoxic or microaerophilic environment, restricting them to less energy-efficient metabolic pathways, resulting in much slower growth and smaller colonies.

The conditions—vacuum-packing (anaerobic), refrigeration (psychrotrophic), and a cured meat environment—select for a specific type of microbe. Lactic Acid Bacteria (LAB) are often facultative or obligate anaerobes, are salt-tolerant, and many are psychrotrophic (can grow at refrigeration temperatures). Certain strains of LAB are heterofermentative, producing lactic acid (sour smell) as well as CO2 gas (bulging). Pseudomonas are obligate aerobes. Molds are aerobic. Thermophilic Clostridium would not grow at 4°C.

MacConkey agar is both selective and differential. Its bile salts and crystal violet inhibit the growth of most Gram-positive bacteria, so the growth itself suggests the bacteria are Gram-negative. The differential component is lactose and a neutral red pH indicator. Bacteria that ferment lactose produce acid, which lowers the pH and causes the colonies and surrounding agar to turn pink/red. Colorless colonies indicate that the bacteria grew (i.e., are likely Gram-negative) but did not ferment lactose, leaving the pH unchanged.