Bacterial Growth and Division

Microbial Growth

• Medical devices that are inserted into a patient's body often become contaminated with a thin biofilm of microorganisms enmeshed in the sticky material secreted by the microorganisms.
• An electron micrograph shows round cells of Staphylococcus aureus bacteria attached to layers of extracellular substrate on the inside walls of an in-dwelling catheter.
• A garbage can served as a rain collector, with a green biofilm on the sides of the container.

Binary Fission

• Binary fission in bacteria begins with DNA replication as the cell elongates.
• A division septum forms in the center of the cell during binary fission.
• Two daughter cells of similar size form and separate, each receiving a copy of the original chromosome through binary fission.
• FtsZ proteins assemble to form a Z ring anchored to the plasma membrane.
• The Z ring pinches the cell envelope to separate the cytoplasm into two new cells.

Bacterial Cell Division

• The parental cell divides and gives rise to two daughter cells.
• Each of the daughter cells divides, giving a total of four cells in the second generation.
• In the third generation, there are eight cells.
• Each division doubles the number of cells.

Bacterial Growth Curve

• The growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a function of time.
• The graph is divided into four phases: lag, log, stationary, and death.
• During the log phase, there is an exponential increase in the number of living bacterial cells.
• During the stationary phase, there is a plateau in the number of living bacterial cells, with the rate of cell division and death being roughly equal.
• During the death phase, there is an exponential decrease in the number of living bacterial cells.

Log Phase Population Growth

• Both arithmetic and semilogarithmic graphs illustrate population growth during the log phase.
• For a bacterial sample, the initial population is presumed to be one cell and with a doubling time of 1 hour.
• When plotted on an arithmetic scale, the growth rate resembles a curve.
• When plotted on a semilogarithmic scale, the growth rate appears linear.

Chemostat

• A chemostat includes a culture vessel fitted with an opening to add nutrients (feed) and an outlet to remove contents (effluent).
• Chemostats effectively dilute toxic wastes and dead cells.
• The addition and removal of fluids is adjusted to maintain the culture in the logarithmic phase of growth.
• When growing aerobic bacteria, suitable oxygen levels are maintained in a chemostat.

Petroff-Hausser Chamber

• The Petroff-Hausser chamber is a specialized slide designed for counting bacterial cells in a measured sample volume.
• A grid is etched on the slide to facilitate precision in counting.
• The grid is made up of squares of known areas.
• The coverslip is 0.2 mm above the grid, and the square containing a bacterial cell count has an area of 0.04 mm².
• If there are 10 cells in the square, the bacterial density is 1.25 million cells/mL.

Fluorescence Staining

• Fluorescence staining differentiates between viable and dead bacterial cells in a sample for counting purposes.
• Viable cells are stained green, whereas dead cells are stained red.

Coulter Counter

• A Coulter counter is an electronic device that counts cells.
• It measures the change in resistance in an electrolyte solution which takes place when a cell passes through a small opening in the inside container wall.
• A detector automatically counts the number of cells passing through the opening.

Serial Dilution

• Serial dilution involves diluting a fixed volume of cells mixed with a dilution solution.
• The previous dilution is used as an inoculum.
• The result is dilution of the original culture by an exponentially growing factor.

Pour Plate Method

• The sample is mixed in liquid warm agar (45–50 ºC) in the pour plate method of cell counting.
• This is poured into a sterile Petri dish and further mixed by swirling.
• This process is repeated for each serial dilution prepared.
• The resulting colonies are counted and provide an estimate of the number of cells in the original volume sampled.

Spread Plate Method

• The sample is poured onto solid agar and then spread using a sterile spreader in the spread plate method of cell counting.
• This process is repeated for each serial dilution prepared.
• The resulting colonies are counted, providing an estimate of the number of cells in the original volume samples.

Most Probable Number Method

• Sets of five lactose broth tubes are inoculated with different volumes of pond water: 10 mL, 1 mL, and 0.1mL.
• Bacterial growth is assessed through a color change in the broth from red to yellow as the bacteria ferments lactose.

Spectrophotometer

• Spectrophotometry is used to measure the turbidity of a bacterial cell suspension as an indirect measure of cell density.
• A spectrophotometer splits white light from a source into a spectrum, allowing the selection of a light wavelength to use for measurement.
• The optical density (turbidity) of the sample depends on the wavelength. It has to be used consistently after it’s chosen.
• Filtered light is passed through the sample, or, a control with only a medium; the light intensity is then measured by a detector.
• In a bacterial suspension, cells scatter the light so that a fraction of the light never reaches the detector.

Cyanobacteria and Budding Bacteria

• Filamentous cyanobacteria replicate by fragmentation.
• Cells of Gemmata obscuriglobus are budding.
• The larger cell is considered the mother cell.
• Nucleoids and the still-forming nuclear envelope of the daughter cell can be seen on electron micrographs.

Biofilm Formation

• Biofilm formation and its life cycle occurs in stages.
• The stages include reversible attachment of planktonic cells, becoming irreversibly attached, growth and cell division, EPS production and water channel formation, and secondary colonizer attachments.
• The initial reversible attachment happens in seconds.
• The irreversible attachment can occur in seconds or minutes
• Growth and cell division occurs over hours and days.
• EPS production and water channel formation occur over hours and days.
• Attachment of secondary colonizers occurs over days and months.

Quorum Sensing

• Short peptides in gram-positive bacteria and N-acetylated homoserine lactones in gram-negative bacteria act as autoinducers.
• Autoinducers function in quorum sensing processes and mediate coordinated responses of bacterial cells.
• The R side chain of the N-acetylated homoserine lactone is specific for species of gram-negative bacteria.

Anaerobic Environments

• Anaerobic environments exist, such as bogs with undisturbed dense sediments devoid of oxygen.
• The rumen (first compartment of a cow's stomach) is an oxygen-free incubator for methanogens.
• The rumen also serves as an oxygen free incubator for obligate anaerobic bacteria.

Bacterial Oxygen Requirements

• Obligate aerobes require oxygen.
• Obligate anaerobes cannot tolerate oxygen.
• Facultative anaerobes can grow with or without oxygen.
• Aerotolerant anaerobes can tolerate oxygen but do not use it.
• Microaerophiles require a limited amount of oxygen.

Anaerobic Jar

• An anaerobic jar holds Petri plates supporting cultures.
• The openings in the side of an anaerobic box are sealed by glove-like sleeves for handling cultures inside the box.

Anaerobe C. perfringens

• Dead tissue accumulating in ulcers provides an ideal growth environment for the anaerobe C. perfringens.
• C. perfringens is a causative agent of gas gangrene, an infection that is worsened in anaerobic conditions.

Catalase Test

• The catalase test detects the presence of the enzyme catalase by noting whether bubbles are released when hydrogen peroxide is added to a culture sample.

Blood Agar Test

• A blood agar test is useful to identify hemolysis.
• A sample blood agar test indicating beta-hemolysis would show that complete lysis of red blood cells had occurred.

Bacterial Motility Test

• A bacterial motility test showing both positive and negative results, shows whether or not the inoculate is motile.
• In a positive result, there would be cloudiness throughout the medium surrounding the inoculation.

Bacterial pH Preferences

• Lactic acid bacteria thrive at a pH close to 4.0.
• Sauerkraut and dishes such as pico de gallo owe their tangy flavor to their acidity.
• Acidic foods have been a mainstay of the human diet for centuries because most microbes that cause food spoilage do not tolerate acidity well.

Prokaryote pH Ranges

• Prokaryotes have different ranges of pH-specific growth patterns.
• Each prokaryote curve has an optimal pH and extreme pH values at which growth is much reduced.
• Most bacteria are neutrophiles and grow best at near-neutral pH.
• Acidophiles have optimal growth at pH values near 3.
• Alkaliphiles have optimal growth at pH values above 9.

Survival at Low pH in the Stomach

• Helicobacter pylori is a corkscrew-shaped bacterium is the real cause of most peptic ulcers.
• H. pylori was identified and isolated by Barry Marshall and Robin Warren and was awarded the Nobel Prize in Medicine.
• H. pylori creates a microenvironment in which the pH is nearly neutral in the stomach.
• H. pylori achieves a microenvironment by producing large amounts of the enzyme urease.
• H. pylori in the stomach rapidly breaks down the urea, producing radioactive CO₂ which can be detected in the patient's breath.

Alkaliphilic Microbes

• Lake Natron in Tanzania is highly alkaline.
• The pink color of the lake is due to the pigmentation of the extreme alkaliphilic and halophilic microbes that colonize the lake.

Sea Vents

• Sea vents provide an extreme environment that is rich in hydrothermal chemicals.
• Macroscopic life (the red tubeworms) is supported by an abundant microbial ecosystem.

Bacterial Growth and Temperature

• Growth rate of bacteria as a function of temperature is commonly skewed towards the optimum temperature.
• The skewed temperature is thought to reflect the denaturation of proteins as temperature rises past the optimum for growth.

Algal Blooms

• Heavy rains can cause runoff of fertilizers into Lake Erie which then triggers extensive algal blooms.

Saline Lake Algae

• The purple color of the Great Salt Lake in Utah is caused by high density of the alga Dunaliella and the archaean Halobacterium spp.

MacConkey Agar

• The lactose-fermenter, E. coli colonies are bright pink on MacConkey agar plates.
• Serratia marcescens, which does not ferment lactose, forms a cream-colored streak on the tan medium.

Gram Positive Cocci

• Gram-positive cocci in clusters.

Listeria and Pregnancy

• A blood sample indicates Jeni, a pregnant woman, has symptoms are due to listeriosis.
• Listeriosis, caused by L. monocytogenes, is a serious infection with a 20% mortality rate.
• Listeria may be transmitted to the fetus.
• Jeni received a high dose of two antibiotics intravenously for 2 weeks for this condition.

Listeria monocytogenes Traits

• L. monocytogenes is a gram-positive short rod found in soil, water, and food.
• It is classified as a psychrophile and is halotolerant.
• It can multiply at refrigeration temperatures (4-10 °C).
• Listeria has a tolerance for high concentrations of salt (up to 10% sodium chloride [NaCl]).
• This is frequently a source of food poisoning.
• Because Listeria can infect animals, it often contaminates food such as meat, fish, or dairy products.
• Listeria contamination is traced to persistent biofilms that form on manufacturing equipment that is not sufficiently cleaned.

Listeria and Progesterone

• Listeria infection is relatively common among pregnant women because of elevated levels of progesterone downregulate the immune system.
• The pathogen can cross the placenta and infect the fetus, often resulting in miscarriage, stillbirth, or fatal neonatal infection.
• Pregnant women are advised to avoid consumption of soft cheeses, refrigerated cold cuts, smoked seafood, and unpasteurized dairy products.
• Because Listeria bacteria can easily be confused with diphtheroids caution is crucial during Listeria identification.

Questions cover bacterial contamination, FtsZ proteins in cell division, growth curve phases, exponential growth calculations, growth on a semilogarithmic scale, characteristics of the lag phase, reasons for growth cessation and binary fission.