Micro: Microbial Growth

Chapter 6. Microbial Growth

• Requirements for microbial growth:

– Two subgroups physical and chemical.

– Physical include: Temperature, pH and osmotic pressure.

– Chemical include: Sources of carbon, nitrogen, oxygen, hydrogen, sulfur, phosphorous and trace elements.

• Physical requirements:

– Temperature- most microorganisms grow at temperatures favored by humans.

• Three temperature classifications.

– Psychrophiles cold loving microbes. Many won’t grow at room temp.

– Example?

– Mesophiles moderate-temperature-loving microorganisms.

–

• Microbes grow in a small range of temps. their maximum and minimum temps. Often vary by only 30^o C.

• Every bacterial species grows at a particular minimum, optimum and maximum growth temperature (Fig 6.1).

• Some archea grow at extremes like hypertermophiles.

– pH most bacteria grow best near neutral 6.5à7.5.

• This is why pickles and sauerkraut are protected because most bacteria can not survive in that pH.

– How are they made?

– Used in science.

– How do you combat this problem?

• However acidophiles can grow in acidic environs including some at a pH of 1.

• Fungi tend to live in slightly acidic environs pH 5-6.

– Ex: Saccharomyces cerevisiae.

– Osmotic Pressure: microbes need a certain osmotic pressure to maintain integrity and get nutrients.

• If a microbe is in a hypertonic solution (what is this?) water will leave the cell and the cell will shrink.

– Plasmolysis is a shrinkage of the cell plasma membrane (Fig. 6.4).

• Extreme halophiles on the other hand require high salt concentrations to survive.

–

– Facultative halophiles are able to grow in high-salt conditions up to 2% some even up to 15%.

– Chemical Requirements:

• Carbon is the structural backbone of living matter and necessary for all organic compounds.

– Chemoheterotrophs get most of their carbon from the source of their energy; proteins, carbohydrates and lipids.

– Chemoautotrophs get their carbon from CO₂.

• Nitrogen, sulfur and Phosphorous.

– DNA synthesis needs nitrogen and phosphorous.

– Protein synthesis needs nitrogen and some sulfur.

– ATP needs phosphorous.

– Nitrogen makes up about 14% of the dry weight of the cell.

– Some bacteria use Nitrogen fixation to get usable nitrogen this can be done as a free living organism or as in symbiosis.

» Ex: Roots of legumes like soybeans, beans and peas.

– Trace elements are very small amounts of certain elements that are needed.

• Ex: Iron, copper, and zinc.

– Oxygen a necessity of life or a poison (Table 6.1)?

• Oxygen is poisonous at high concentrations and is often combined with hydrogen to form water neutralizing the poison.

• Some microbes do use molecular oxygen.

–

» Tough since water doesn’t dissolve well in water or in many environments.

– So many organisms are facultative anaerobes.

» Can grow in the absence or presence of oxygen.

» Ex: E. coli and most importantly yeast.

– Obligate anaerobes cannot use molecular oxygen and most are harmed by it.

– Harmful oxygen includes H₂O₂, Superoxide (O₂^-) and hydroxyl radical (OH^.).

– Many obligate anaerobes don’t have the enzymes to deal with these reactive oxygen species (ROS).

– Aerotolerant anaerobes cannot use oxygen but tolerate it well.

– Microaerophiles only grow in low concentrations of oxygen.

– Organic growth factors directly obtained from the environment.

• Ex = vitamins

• Culture media is nutrient material prepared for microorganism growth.

– Microbes introduced into a culture are an inoculum.

– Microbes that grow in or on a culture are referred to as a culture.

• Culture media must be sterile.

•

– Agar is a complex polysaccharide made from a marine algae.

• Used in jellies and ice cream.

• Important properties: microbes cannot degrade it, liquefies at 100^oC and solidifies at 40^oC.

– Chemically defined medium is one in which the exact chemical composition is known.

• Used for laboratory experimental work or autotrophic bacteria.

– Complex media is made up of nutrients from extracts of yeasts, meat, plants or digests of protein.

• Nutrient broth and nutrient agar.

– Anaerobic growth Media and Methods.

• Must use reducing media that contain chemicals like sodium thioglycolate that combine with oxygen to deplete it.

• If you need to grow an anaerobe on a plate a anaerobic jar may be used (Fig 6.5).

– Special culture techniques.

• Some organisms cannot be cultured easily on laboratory media.

– Example: Mycobacterium leprae cultured in armadillos.

– Labs may have special incubators for anaerobes and capnophiles (microbes that grow better with increased CO₂) (Fig 6.7).

» CO₂ candle jars.

– Selective and differential media are used to detect the presence of specific microorganisms.

• Selective media are used to suppress the growth of unwanted bacteria and encourage the growth of desired microbes.

• Example: Sabourad’s dextrose agar which has a pH of 5.6 is used to isolate fungi because of pH.

• Differential media make it easier to distinguish between colonies of the desired organism from other colonies.

– Example: Streptococcus pyogenes the bacteria causes lysis of the blood cells in blood agar and makes a ring around the cells.

– Enrichment media is usually a liquid media and provides nutrients and environmental conditions that favor the growth of a particular microbe.

• To increase the number of a microbes to prevent missing a microbe that may be in small numbers.

• Often used on soil or fecal samples.

• Example: soil sample looking for bacteria that grow on phenol. Culture à Move à Grow àMove à Grow Growth is the enrichment stage.

• Obtaining pure Cultures!!!!

– Most starting materials contain many organisms.

•

• If these materials are plated out they will form colonies that are exact copies of the original organism.

• A visible colony arises from a single spore or vegetative cell or from a group of the same organism attached to one another in clumps or chains.

• Most bacteriological work requires pure colonies.

• One way to recover pure single colonies is the streak plate method (Fig 6.10).

– Doesn’t work well when the numbers of that cell type are low.

– Needs selective enrichment first if cell number is low.

• Preserving bacterial cultures.

– Deep-freezing is a process by which a culture is placed in suspension medium and frozen at temperatures between –50^oC and –95^oC.

• Most commonly done at ~-80^oC in ultra-low freezers.

• Suspension media is often Nutrient broth and 15% glycerol.

– Lyophilization (freeze-drying) the organism is quik frozen as above and dried under a vacuum.

• Bacterial culture growth: Analyzing the growth of bacterial cultures is an essential part of bacteriology.

– Bacterial division: Bacterial growth refers not to the increase in size of cells but the increase of numbers of cells.

•

• A few bacteria reproduce by budding.

– Generation time- the time required for a cell to divide (Fig 6.12).

• When the number of cells in each generation is expressed as the power of 2 the exponent tells the number of doublings (generations) that have occurred.

– Doubling time can be as low as 20 minutes (E. coli) and as high as 24 hours or more.

– Most 1-3 hours.

– After 20 generations a single cell of E. coli would be over 1,000,000.

» 30 generations = 10 hours = 1,000,000,000

– Difficult to graph such high numbers that is why logarithmic scales are used, bacterial growth curve (Fig 6.13).

• Logrithmic representations are much easier to graph and is necessary for proper understanding of microbial populations.

– Phases of Microbial Growth: When a few bacteria are inoculated into a liquid and the population counted at intervals it is possible to plot a bacterial growth curve.

• Four Basic Phases of Growth: Lag phase, Log Phase Stationary phase and death phase (Fig 6.14).

•

– Period of intense metabolic activity including DNA and enzyme sythesis.

• The log phase or exponential growth phase- cellular reproduction is at its most active.

– generation time equals a constant minimum rate that is why you see a straight line.

– Microbes are at their most sensitive here many antimicrobial agents work best during this phase by interfering with growth (Penicillin).

• Stationary phase is when cell death equals new cells and the metabolic activity of cells decline.

– Can be combated by replacing spent medium with fresh medium.

• Death phase or logarithmic decline occurs when deaths outnumber new cells.

– Direct Measurement of Growth.

• Microbial populations are usually measured by cell number per milliliter or by population mass.

• Because of the incredible numbers of cells in a bacterial population they are usually measured indirectly or directly in small sample sizes.

• Dilutions are done to count cells more easily.

• Plate counts: Assumes that each living cell produces a single colony.

– Some however form from chains or clumps and are expressed as colony forming units.

– Serial dilutions down to approximately < or = 250 cells are best (Fig. 6.15).

• Pour plates and spread plates (Fig 6.16).

– Pour plates have colonies suspended in the medium

» Can be hard to visualize.

– Spread plates have the cells spread across the surface of a solidified medium.

» Usually spread with a bent glass rod.

–

• Filtration- bacteria can be passed through a filter to trap them and then they can be transferred to a petri plate containing appropriate medium.

– Used to recover bacteria out of water.

• Most probable number method- Statistical estimating technique based on the fact that the more bacteria the more dilutions needed to get no bacteria.

– Used most often for bacteria that do not grow on solid media.

• Microscopic count uses a specifically designed Petroff-Hausser cell counter slide to microscopically count the cells in a known volume (Fig 6.19).

• Turbidity- uses a spectrophotometer to estimate the number of cells (Fig 6.20).

– In a spectrophotometer light is transmitted through a dilution of the culture (usually a 1:10 dilution)

– As microbe numbers increase light passing through the culture decreases.

– The output is often absorbance or optical density.

» Absorbance is a logarithmic expression of the amount of light that gets through the culture.

» The number of cells per absorbance unit is a known constant for most microorganisms.

• Example: of turbidity use in cell counting.

– Yeast 1.0 A₆₀₀ unit = 10⁷ cells (10,000,000).

» A₆₀₀= absorbance at a wavelength of 600 nm.

• So if you are reading a 1:10 dilution of your yeast culture and the absorbance from the spectrophotometer reads as 0.1 you have a culture that has an A₆₀₀ of 1.0 and has 10,000,000 cells per milliliter.

– How many cells if the reading was 0.2?

• Metabolic activity can be used to estimate cell number, the amount of some product produced in normal organism activity can be used to estimate cell number.

– CO₂ or acid is directly proportional to cell count.

• Dry weight can be used especially for molds and other filamentous microorganisms.

– Organism is dried and then weighed.

– Not as accurate.