#bacterial culture

Microbial Physiology

Which of the following is not a factor affecting bacterial growth?

Microbial growth influenced by physicochemical variables reflecting physiology. Temperature controls enzyme catalysis activation energy, membrane fluidity, protein thermostability, cardinal minima optima maxima distinct per species. Oxygen availability categorizes obligate aerobes requiring cytochrome oxidases and superoxide dismutase, obligate anaerobes lacking detoxification killed by oxygen, facultative anaerobes switching via Fnr ArcAB, microaerophiles needing low oxygen. pH affects proton motive force, amino acid charge, solubility nutrients. Water activity aw controlled by osmotic solutes, halophiles adapt via compatible solutes ectoine trehalose and modified proteome rich in acidic residues. Light essential only for phototrophs using bacteriochlorophyll, not major factor for chemoheterotrophs cultured in dark incubators. Presence of mitochondria is irrelevant because bacteria are prokaryotes lacking membrane bound organelles, their energy conservation across plasma membrane via electron transport chain composed of NDH dehydrogenases quinones cytochromes, proton extrusion forming gradient driving F1Fo ATP synthase. Mitochondria originate from alphaproteobacterial endosymbiosis in eukaryogenesis, not component of bacterial cell. Hence mitochondria absence as factor distinguishes domains.

Ref: Madigan et al., Brock Biology of Microorganisms, 16th ed., Chapter 7: Factors affecting growth - Mitochondria absent.

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Microbial Physiology

A bacterial culture was diluted 1000-fold, and 0.1 mL of this sample was plated. If 100 colonies formed, what was the or

Viable count estimation via plate counting requires accounting for dilution magnitude and volume plated to infer original concentration. Only 30 to 300 colonies per plate statistically reliable. General formula colony forming units per mL equals colonies observed divided by product dilution factor fraction and volume plated in milliliters. If culture diluted 1000 fold meaning dilution factor 1 times ten to minus three, and 0.1 mL aliquot plated yielding 100 colonies, denominator equals ten to minus three times 0.1 equals ten to minus four. One hundred over ten to minus four equals one million per mL in diluted tube. Multiplying back by dilution factor 1000 gives original at one million? Actually careful: colonies already from diluted sample, original calculated as colonies over volume times inverse dilution equals 100 over 0.1 times 1000 equals 1 times ten to six. This illustrates how small pipetting error amplifies hundredfold. Mastery of this calculation central for food safety standards, water coliform counts, antibiotic minimal inhibitory concentration assays where accurate CFU essential for regulatory compliance, dose response, and reporting results as CFU per mL or per gram.

Ref: Prescott's Microbiology, 11th ed., Chapter 7: Viable count - CFU/mL = colonies/(dilution×volume).

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Microbial Physiology

What is the primary reason for an extended lag phase in bacterial cultures?

Extended lag phase not caused by high density per se but by requirement for extensive macromolecular synthesis before replication can resume. Cells transferred from old, stationary, or differently composed medium lack enzymes for new carbon source utilization, transport permeases, central metabolic dehydrogenases, and contain degraded ribosomes, oxidized proteins, and damaged DNA. They must induce transcription of catabolic operons lac, mal via cAMP CRP, synthesize ribosomal proteins rps, rpl, produce tRNA, generate ATP via substrate level phosphorylation, repair chromosome via Uvr, Rec systems. If transferred to medium requiring enzymes absent previously, time required for de novo mRNA translation lengthens lag. Cold shock, osmotic shock, antibiotic exposure exacerbate lag as chaperones GroEL DnaK and proteases Clp needed. Conversely high inoculum quorum signals like autoinducer 2 can shorten lag by sharing metabolites. Therefore requirement for enzyme synthesis and repair explains why lag can last hours while population numbers appear unchanged despite intense metabolic remodeling inside cells preparing for division.

Ref: Prescott's Microbiology, 11th ed., Chapter 7: Extended lag - Enzyme synthesis requirement.

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Microbial Physiology

The bacterial growth curve represents the number of live cells over time when cultured in:

Laboratory bacterial growth curve plotting viable number versus time is obtained from synchronous population in closed liquid batch culture, typically nutrient broth shaken for aeration. Solid agar shows colonies spatially separated, diffusion limited, colony counts reflect single time point not temporal kinetics. In broth, aliquots removed at intervals, optical density at 600 nm measures light scattering correlated to biomass and serial dilutions plated counting colony forming units representing live cells. Curve reveals four phases lag adaptation, log exponential where nutrients exceed Ks Monod constant, stationary zero net growth, death decline. Liquid allows uniform distribution of nutrients, oxygen, quorum molecules acyl homoserine lactones, ensures representative sampling. Continuous culture chemostat extends exponential by fresh medium inflow. Solid medium suited for isolation and enumeration but not for dynamic growth modeling. Recognizing that growth curves derive from liquid batch culture is essential for interpreting specific growth rate, carrying capacity, yield coefficients in physiology experiments, antibiotic time-kill curves, and industrial fermentation scale up where broth conditions are strictly controlled.

Ref: Brock Biology of Microorganisms, 16th ed., Chapter 6: Bacterial growth curve - Batch liquid culture.

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Microbial Physiology

The CFU/mL of a bacterial culture is calculated using the formula:

Colony forming units per milliliter provides standard measure of viable culturable bacteria assuming each colony derives from single viable unit capable of growth on agar. To achieve statistically reliable counts, serial tenfold dilutions are prepared in diluent and fixed aliquot such as 0.1 mL is spread onto nutrient agar via spread plate using sterile spreader or mixed into molten agar via pour plate. After appropriate incubation colonies are counted; ideal range 30 to 300 avoids overcrowding and sampling error. Because only diluted fraction was plated, conversion to original density requires normalization. Correct formula CFU per mL equals number colonies counted multiplied by total dilution factor reciprocal of dilution plated for example ten to six for ten to minus six dilution divided by volume plated in milliliters. Illustration 50 colonies on ten to minus five dilution plated 0.1 mL yields 50 times ten to five over 0.1 equals five times ten to seven CFU per mL. Omitting volume division underestimates by tenfold, dividing rather than multiplying by dilution factor inverts scaling leading to erroneous infectious dose, growth rate and regulatory compliance assessment in food and pharmaceutical microbiology quality control.

Ref: Prescott's Microbiology, 11th ed., Chapter 6: CFU/mL Calculation and Dilution Factor Method.

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