culture medium n. A liquid or gelatinous substance containing nutrients in which microorganisms or tissues are cultivated for scientific purposes.

(1) Chapter Title: Growth and Culturing of Bacteria (2) (2) Microbial growth (a) (a) "Because individual cells grow larger only to divide into new individuals, microbial growth is defined not in terms of cell size but as the increase in the number of cells, which occurs by cell division." (b) (b) This emphasis has practical application since it is typically far easier to measure increases in cell number than it is to measure increases in cell size (c) (c) Furthermore, unless cell division is synchronized, cells will typically vary in size across an even homogeneous population, thus making measurement of cell size almost irrelevant as a means of measuring microbial growth (3) Binary fission (d) (a) The majority of bacteria reproduce by a mechanism termed binary fission (e) (b) Binary fission is much simpler than the mechanisms of cell division seen in eucaryotic cells (f) (c) See Figure 6.1, Binary fission (g) (d) [binary transverse fission] [binary fission (nice cartoon illustration (h) (e) (if there are two fish in a lake, and one of them is dead, thats called binary fishin) (i) (f) binary fission (4) Tetrad (j) (a) Tetrads are a cell arrangement that is a consequence of binary fission not resulting in complete separation of cells, and that occurs in two planes, thus producing a square consisting of four cocci, one at each corner (5) Sarcinae (k) (a) Sarcina are a cell arrangement that is a consequence of binary fission that does not result in complete separation of cells, and that occurs in three planes, thus producing cubes consisting of eight cocci, one coccus at each corner (3) (6) Standard bacterial growth curve (a) (a) Bacteria added to fresh media typically go through four more-orless distinct phases of growth (i) (i) Lag phase (A) (ii) (ii) Log (logarithmic or exponential) phase (B) (iii) (iii) Stationary phase (C)

(b)

(iv) (b)

(iv)

Decline (death) phase (D)

(d) See Figure 6.3, A standard bacterial growth curve (4) (7) Lag phase (a) (a) Transfers of bacteria from one medium to another, where there exist chemical differences between the two media, typically results in a lag in cell division (b) (b) This lag in division is associated with a physiological adaptation to the new environment, by the cells, prior to their resumption of division (c) (c) That is, cells may increase in size during this time, but simply do not undergo binary fission (5) (8) Log phase (logarithmic phase, exponential phase) (a) (a) Lag phase is followed by log phase during which binary fission occurs (b) (b) This phase of growth is called logarithmic or exponential because the rate of increase in cell number is a multiplicative function of cell number (c) (c) This can be seen in a graph of cell number versus time where cell numbers increase at ever increasing rates with time or generation; that is, the rate of increase is a function of absolute cell number such that the more cells present, the faster the population of cells increases in size (at least, during log phase) (d) (d) See Figure 6.4, Nonsynchronous growth (e) (e) When graphed on semi-log graph paper (Figure 6.3, i.e., log cell number versus time), log-phase growth produces a straight line

(6) (9) Continuous culture (serial transfer) (a) (a) A means of keeping cultures in log phase can be accomplished either by employing a chemostat or via serial transfer (b) (b) A chemostat involves adding fresh medium to a culture, mixing, and then allowing an equal volume of culture to drain from the vessel; this is typically done continuously (i.e., a steady stream of fresh medium is added) (c) (c) Serial transfer means taking a volume of culture and diluting that volume into fresh media (7) (10) Generation time (a) (a) Generation time it takes a bacterial population to double in size (number) during log-phase growth (b) (b) Note that the time it takes for the population to double in size does not change with cell number (so long as cells remain in log phase) (c) (c) That is, with exponential growth, the absolute increase in cell number increases as cell number increases while the relative increase remains invariant (d) (d) Typically, generation times range from 20 minutes to 20 hours depending on the bacterial species/strain and the conditions during which log-phase growth is occurring (8) (11) Stationary phase (a) (a) Stationary phase is a steady-state equilibrium where the rate of cell growth (division) is exactly balanced by the rate of cell death (i.e., increase in cell number due to cell divisions exactly balanced by a decrease in cell number due to death) (b) (b) Cell death (or, at least, lack of cell growth) occurs because of a loss of limiting nutrients (due to their incorporation into cells during logphase growth) or a build-up of toxins (due to their release during log-phase growth, e.g., fermentative products) (c) (c) Note that the simplest conditions that will result in a stationary phase is when both the rate of cell increase and the rate of cell death together equal zero (i.e., cells neither die nor are born) (9) (12) Decline phase (death phase) (a) (a) Stationary phase, in a standard bacterial growth curve, is followed by a die-off of cells (b) (b) Cell death in bacteria cultures basically means that the cells are unable to resume division following their transfer to new environments (c) (c) Typically this die-off occurs exponentially, i.e., such that cell number graphed against time, using a semi-log scale for cell number, results in a straight line (i.e., see Figure 6.3) (d) (d) This death occurs because vegetative cells can survive exposure to harsh conditions (few nutrients or too-many toxins) for only so long (10) (13) Solid medium

(a) (b) (c) (d) (e) (f)

(a) Solid media contains agar, which is a compound that goes into water solution at temperatures approaching boiling, and then, once in solution, solidifies the medium at room (<40C) temperature (b) Subsequent exposure to high temperature (i.e., boiling) will melt the medium (c) Exposure to relatively low temperatures (i.e., >40C), however, will not melt the medium, thus allowing incubation of solid medium at various temperatures (compare to gelatin which liquefies at 37C) (d) Once boiled, agar-containing medium will stay liquid at 45C (e) This allows solid medium to be poured into various vessels at temperatures that will not kill most cells (nor melt vessels), followed by a solidification of the medium (f)

(11) (14) Colonies (a) (a) Colonies represent piles of cells descended, assuming pure culture technique and sufficiently few colonies on a single plate, from a single parent cell, all growing on or in a solid medium

(b)

(b)

(c) (d) (e)

(c) The four phases of bacterial growth can be observed within a single colony, with the edges displaying lag and log phases and the interior can display stationary and then decline phases (d) Various colony morphologies (no need to memorize): (e)

(f)

(f)

(12) (15) Pour plate (a) (a) The pour-plate method is employed for bacterial-cell enumeration and isolation (b) (b) In the pour-plate method of addition of cells to solid medium contained within a petri dish, cells are added to melted (but not too hot) solid medium (c) (c) The melted solid medium is then poured into a petri dish and allowed to harden (d) (d) Colonies appear both within, beneath, and on top of the agar (13) (16) Spread plate (a) (a) The spread plate method is employed for bacterial-cell enumeration and isolation (b) (b) In the spread-plate method of addition of cells to solid medium, a small volume of culture is dropped onto the surface of agar that has already hardened in a petri dish (c) (c) The volume is then spread around the agar surface (d) (d) Colonies will grow solely on the surface of the agar (e) (e) This technique is advantageous particularly when cells are sensitive to exposure to relatively high temperatures plus the method does not require a prior melting of the solid medium (14) (17) Standard plate count (a) (a) When bacteria are placed on a solid medium, ideally each colony is founded by only a single bacterial cell (obviously, given clumping and various cell arrangements, this ideal is not always met) (b) (b) Thus, addition of a known quantity of bacterial cells to a solid medium should produce the same number of colonies (c) (c) This result can be employed backward so that the number of colonies grown on solid medium can be used to estimate the number of individual bacteria that were added to the solid medium (d) (d) This number, in turn, may be employed to estimate the concentration of bacteria present in a culture (15) (18) Colony-forming unit (CFU) (a) (a) Whether a single cell or a clump of cells or whatever, what grows into an isolated colony is termed a colony-forming unit (b) (b) When doing standard plate counts, what is being estimated are numbers of colony-forming units (c) (c) The number of colonies that may be counted per petri dish (and therefore the number CFUs that may be enumerated) is limited by statistics (at the low end) and space on the dish (at the upper end) to between 30 and 300 colonies/CFUs per plate

(16) (19) Serial dilution (a) (a) For a given agar surface, there are only so many colonies that may be present before it becomes impossible to accurately count the number of colonies present (b) (b) This number serves as a limit on the concentration of bacteria in cultures that may be enumerated using the standard plate-count technique (c) (c) How to get around this limitation? This may be achieved by diluting cultures before enumerating them (d) (d) By keeping track numerically of the degree of diluting employed, the concentration of bacteria in the pre-diluted culture may be estimated (e) (e) Due to limitations in the size of the volumes that may be conveniently handled (i.e., both very large volumes and very small volumes are difficult to handle), relatively large dilutions are typically handled serially (f) (f) For example, a 10-fold dilution followed by a second 10-fold dilution (i.e., 1 ml from a parent culture diluted to 9 ml diluent followed by mixing followed by 1 ml from the diluted culture diluted to an additional 9 ml diluent followed by mixing) gives a total of a 100-fold dilution (g) (g)

(h) See Figure 6.6, Serial dilution (i) See Figure 6.7, Calculation of the number of bacteria per milliliter of culture using serial dilution (17) (20) Direct microscopic counts (a) (a) A more direct means of enumerating bacteria is done by viewing them through a microscope (b) (b) This method's limitations are that only relatively high concentrations of bacteria may be enumerated and the method cannot distinguish living from dead bacteria

(h) (i)