Supplemental Lecture** (98/04/03 update) by **Stephen T. Abedon** (**abedon.1@osu.edu**)**

- Chapter title: Microbial Growth
- A list of vocabulary words is found toward the end of this document
- Microbial growth
- Microbial growth may be described as occurring in different ways under different circumstances.
- Increase in both population size and population mass:
- Microbial populations tend to increase in number and in cell mass simultaneously.
- Increase in cell number and increase in cell population mass both usually occur in a measurably coordinated fashion, and thus are often used interchangeably, even synonymously (though obviously we must be speaking in terms of populations of cells for this to be true).
- Note that, for bacteria, while the cell population and population mass typically increase with time (with growth), over the course of population growth individual cells actually cycle through increases and decreases in cell mass (i.e., growth, division, growth, division, growth . . .)
- Bias toward cell number:
- When a microbiologist speaks of microbial growth it is usually increase in cell number that she is after.
- The reason for this bias is that a typical microbiologist is more interested in population characteristics than in the characteristics of individual cells, or both (since the characteristics of individual cells tend to be studied, by necessity, within the context of populations of cells).
- Consequently, there is a tendency for microgiologists to follow microbial growth as populations rather than following the growth of individual cells, and therefore microbiologists tend to be more interested in population sizes than the size (mass) of any indvidual cell. Futhermore, the typical measurement of microbial growth will be done over the span of more than one microbial generation.
**Increase in cell number**- An
*increase in cell number*is an immediate consequence of cell division. - Because most bacteria grow by binary fission, doubling in cell number usually occurs at the same rate that individual cells grow and divide.
**Increase in cell mass**- Doubling in size:
- Individual cells of many species double in size between divisions.
*Cell mass*thus increases at the same rate as cell number.- The implication of this is that while increase in cell number may be emphasized while considering microbial growth, increases (and decreases) in individual cell masses are also occurring, though these increases and decreases ballance each other out such that the average cell size tends to remain constant under constant conditions.
- Anabolic process:
- The increase in mass is a consequence of anabolism.
- For anabolism to occur a cell must be situated in an environment that supplies all necessary nutrients and which physically falls into a range in which growth can occur.
**Binary fission**- Procaryotic cell division:
*Binary fission*is the process by which most procaryotes replicate.*Binary fission*generally involves the separation of a single cell into two more or less identical daughter cells, each containing, among other things, at least one copy of the parental DNA.- Stepwise process:
- The first steps of
*binary fission*include cell elongation and DNA replication. - The cell envelope then pinches inward, eventually meeting.
- A
*cross wall*is formed and ultimately two distinct cells are present, each essentially identical to the original parent cell. - See illustration below.
**Illustration, binary fission****Generation time****[doubling time]**- Procaryotic cell division:
- A bacterial
*generation time*is also know as its*doubling time*. *Doubling time*is the time it takes a bacterium to do one binary fission starting from having just divided.- And ending at the point of having just completed the next division.
*Generation times*vary with organism and environment and can range from 20 minutes for a fast growing bacterium under ideal conditions, to hours and days for less than ideal conditions or for slowly growing bacteria.**Standard bacterial growth curve**- The
*standard bacterial growth curve*describes various stages of growth a pure culture of bacteria will go through, beginning with the addition of cells to sterile media and ending with the death of all of the cells present. - The phases of growth typically observed include:
- lag phase
- exponential (log, logarithmic) phase
- stationary phase
- death phase (exponential or logarithmic decline)
- In standard bacterial growth curves one keeps track of cell growth by some measure or estimation of cell number.
**Exponential [log or logarithmic] growth (phase)**- Back-to-back divisions:
*Exponential growth*is a physiological state marked by back-to-back division cycles such that the population doubles in number every generation time.- Note that during
*exponential growth*there is no change in*average*cell mass, though individuals cells are constantly changing in mass as they increase in mass, then divide thus rapidly decreasing in mass (while increasing in number). - The algebra of exponential growth:
- Note that during
*exponential growth*the number of cells present at any given time is a multiplicative function of the number of cells present at a previous time. - Under constant conditions the multiplicative increase in cell number consequently is constant for any given interval of the same duration.
- Examples:
- If a
*log phase*culture goes from 2 cells to 4 cells during a 20 minute interval, then the culture will go from 4 cells to 8 cells during the next 20 minutes. - If a
*log phase*culture goes from 2 cells to 6 cells during a 60 minute interval, then the culture will go from 6 cells to 18 cells during the next 60 minutes. - If during
*exponential phase*there are 10 cells present at time 0, and 100 cells present at time 200, then at time 400 there will be 10,000 (100 * 100) cells present. **Vegetative cell**- A
*vegetative cell*is one which is capable of actively growing. - Constrast with endospore.
**Lag phase**- Lag in division:
- Upon a change in environment (especially from a rich environment to a poor environment), or when going from stationary phase to exponential phase, there is a lag before division resumes.
- For example, stationary phase
*Escherichia coli*placed in an excess of sterile broth will go through a*lag phase*during which they increase in cell size but do not divide. They will divide only once they have reached the size of a cell which is about to divide during exponential growth under those conditions. - During this time a culture is said to be in
*lag phase*. - Increase in mass:
- During
*lag phase*cells increase in mass but do not divide. - In other words, there is no change in number, but an increase in mass.
- "The length of the lag phase is determined in part by characteristics of the bacterial species and in part by conditions in the media---both the medium from which the organisms are taken and the one to which they are transferred. Some species adapt to the new medium in an hour or two; others take several days. Organisms from old cultures, adapted to limited nutrients and large accumulated wastes, take longer to adjust to a new medium than do those transferred from a relatively fresh, nutrient-rich medium." (p. 138, Black, 1996)
**Stationary phase***Stationary phase*is classically defined as a physiological point where the rate of cell division equals the rate of cell death, hence viable cell number remains constant.- No cell division:
- Note that when cell division = 0 and cell death = 0, then the rate of cell division = rate of cell death.
- In other words, when cells stop dividing but have not yet started dying they are in
*stationary phase*. - A way to distinguish these possibilities is to compare viable count with total count.
- If both total counts and viable counts don't change then you know that there is both no cell division and no cell death.
- If total count increases while viable counts remain constant, then you know that you are observing a true balance between ongoing cell division and cell death.
- Physiological adaptation to cell excess:
- Stationary phase usually occurs when cell concentration is so great and that some aspect of the environment is no longer able to serve the requirements of exponential growth.
- Stationary phase is a time of significant physiological change and particularly involves the physiological adaptation of cells to survival through periods of little growth.
**Cell death**- In single celled microorganisms
*cell death*is the point at which reinitiation of division is no longer possible. - Qualified definition:
- Note that the concept of
*cell death*is actually dependent on how one attempts to reinitiate growth. - Particularly, there are ways to gently
*revive*some microbes from physiological states that would result in permanent lack of growth in other growth environments. - An analogous situation would be a person with an injury that is inevitably fatal in a third-world hospital, but readily treated in a first-world hospital.
- Example: seeds:
- Another analogy is with a plant seed. You can try to sprout it in all kinds of environments but not all will work out in the seed's favor. You may end up killing the seed by allowing it to attempt to germinate in the wrong environment.
- The more degraded is the seed prior to planting, the greater the likelihood that germination will not successfully occur unless you take great care to make sure sprouting conditions are as close to ideal as you can make them.
**Death phase****[logarithmic decline, exponential decline]***Death phase*is a physiological point at which cell deaths exceed cell births.- More specifically, viable count declines.
- "During the
*decline phase*, many cells undergo*involution*---that is, they assume a variety of unusual shapes, which makes them difficult to identify." (p. 140, Black, 1996) **Endospore****[spore, sporulation, sporogenesis, activation, germination]**- Tough, dormant state:
- A very tough, dormant form of certain bacterial cell that is very resistant to desiccation, heat, and a variety of chemical and radiation treatments that are otherwise lethal to non-
*endospore*bacterial cells. - At least part of the toughness associated with a
*spore*is found in its very tough outer layers, called a coat. - Only some bacteria produce
*endospores*. *Endospores*of some bacteria can last so long under proper conditions that various*endospores*found in such things as Egyptian mummies are likely the oldest living things.- Sporulation and sporogenesis:
*Sporulation*and*sporogenesis*refer to the formation of*endospores*by*vegetative*(i.e., growing) cells.- The
*endospore*is actually the intracellular product of*sporogenesis*. - A
*spore*is an endospore which has been released from a cell, i.e., it exists is a free state. - In bacteria the formation of a
*spore*is not considered to be an act of reproduction. Indeed, the formation of the*endospore*is directed by the DNA that will ultimately be found in the*spore*, and the sister DNA found in the vegetative part of the cell ultimately is destroyed. - The first step of germination, often requires some kind of coat traumatizing insult such as high temperature or low pH.
- The transformation from the
*endospore*state to the vegetative state. - The key thing to worry about with
*endospores*is that they are capable of*germinating*despite harsh treatment, and thus can potentially produce actively replicating cells where there may have been none previously prevent. - Of those bacteria on your list, the following are spore formers (note that all are gram-positives):
**Vocabulary**- Binary fission
- Binary fission, illustration
- Cell death
- Death phase
- Doubling time
- Endospore
- Exponential decline
- Exponential growth
- Exponential phase
- Generation time
- Lag phase
- Log phase
- Standard bacterial growth curve
- Stationary phase
- Vegetative cell
**Practice questions**- Which is the more reasonable temporal order for a culture transferred at some point to fresh medium (circle only on correct answer)? [PEEK]
- exponential decline, stationary phase, exponential phase, lag phase
- stationary phase, lag phase, exponential phase, exponential decline
- exponential decline, stationary phase, exponential phase, lag phase
- exponential phase, stationary phase, exponential decline, lag phase
- all of the above
- none of the above
- If at time 0 you had 10 million cells and two hours later had 40 million cells, what is the generation time? [PEEK]
- In the figure below, during period A, cells are (circle only one correct answer)? [PEEK]
- increasing in number
- decreasing in number
- increasing in mass
- decreasing in mass
- all of the above
- none of the above
- In the figure above, during period B, these cells undergoing binary fission are
*not*displaying (circle only one correct answer)? [PEEK] - a net increase in number
- a net increase in average cell mass
- more cell births than cell deaths
- exponential growth
- all of the above
- none of the above
- If you have a generation time of 20 minutes, if you started with 5 cells, how many cells would you have in 200 minutes (if you want you can display this latter answer using exponents)? [PEEK]
- How does the average cell mass (that's
*average*, not*total*cell mass) of an exponentially growing culture of*Escherichia coli*change over time? (circle correct answer) [PEEK] - increases
- increases slightly
- stays nearly the same
- decreases slightly
- decreases
- all of the above
- none of the above
- A culture contains 17 x 2
^{8}cells. How many cells did that culture contain six generations earlier? (show your work) [PEEK] - If viable count declines but total cell count remains constant, what phase of growth is a culture in? [PEEK]
- lag phase
- exponential phase
- stationary phase
- exponential decline
- all of the above
- none of the above
- A culture displays exponential death when assayed using MacConkey agar but no decline in viable count when assayed using colony count agar. What might you conclude about MacConkey agar relative to colony count agar. (<10 word answer) [PEEK]
- You have a culture with 19 cells in it. The culture goes through three generations. How many cells are now in the culture (assume that the culture is in exponential phase throughout the experiment, only viable cells are being measured and are of any concern to you, and that three generations implies three rounds of replication). [PEEK]
- You have a culture with 400 cells in it. It is going through exponential decline at a rate of 50% die-off per minute. After three minutes, how many viable cells do you have left. [PEEK]
- A tough, dormant, seed-like state exhibited by some Gram-positive bacteria is called _________. (circle one correct answer) [PEEK]
- stationary phase.
- encystation.
- the vegetative state.
- an endoseed.
- all of the above.
- none of the above.
- Circle all of the endospore formers. [PEEK]
*Bacillus anthracis, Bacillus subtilis, Bdellovibrio spp., Bordetella pertussis, Borrelia burgdorferi, Chlamydia trachomatis, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Escherichia coli, Gardnerella vaginalis, Helicobacter pylori, Hemophilus influenzae, Klebsiella*spp*., Lactobacillus*spp*., Legionella*spp*., Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella pestis, Proteus*spp*., Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Salmonella*spp*., Serratia marcescens, Shigella*spp*., Staphylococcus aureus, Streptococcus*spp*., Treponema pallidum, Vibrio cholerae, Yersinia pestis* - How is it that bacterial cells, during binary fission, are able to assure that each resulting daughter cell contains exactly one bacterial chromosome? [PEEK]
- Name (and properly present) the genus (or binomial) of a Gram-positive bacterium whose cells are longer than they are wide and which forms endospores. [PEEK]
- Staring with 10 bacteria, a doubling time of 30 minutes, and given exponential growth, how many bacteria will you have after 3000 minutes (a little over 2 days) of continuous exponential growth? (note: if you find it simpler, let me encourage you to leave your answer in exponential form, i.e., you don't have to multiply it out to the scientific notation form if you don't want to) [PEEK]
- Name the four phases, in order, of a standard bacterial growth curve. [PEEK]
- During both lag phase and log phase, cells display increases in individual cell mass. What is the other aspect of microbial growth that, by contrast, occurs during log phase but not during lag phase? A18
- Define microbial cell death. [PEEK]
- An otherwise typical bacterial cell increases from one cell to 256 cells in 10 hours. What is the generation time of this organism? [PEEK]
**Practice question answers****iv, why? Consider a culture in exponential phase. The culture medium runs out of a limiting substrate and the culture stops growing (stationary phase). Eventually cells start dying (exponential decline). If a sample of these dying cells are transferred to fresh medium the cells can start growing again. However, before they can divide they must first adapt to the new environment, i.e., go through lag phase. Note that the only time you would expect a lack of lag phase is if you transferred exponential phase cells to essentially an identical culture medium (or, of course, if you transfer only dead cells).**- 1 hour, 10 million --1 hour-- 20 million --1 hour-- 40 million.
- iii, they are in lag phase and therefore are increasing in mass but not increasing in number. You know there is no change in cell number because the graph says so. If the cell number value is a viable count then you could also be certain that there is no decrease in cell number. In fact, since the figure shows a decline phase you can be reasonably confident that if a decrease in cell number were occurring during phase "A" you would see it. Finally, you only know that the cells are increasing in mass because you have memorized what occurs during the various phases of growth and phase "A" is clearly lag phase.
- ii, this is a tricky question. The key word is average. There is no net change in average cell mass during exponential growth. This is not to say that there is no change in the mass of individual cells. It is just that cells increase in mass to the point of doubling, then divide thus cutting their mass in half. Over the entire population this works out to no change in average cell mass during exponential phase.
- 5 cells x 2 for every generation. 200 minutes of 20 minutes generations is clearly 10 generations. Therefore 5 x 2 x 2 x 2 x 2 x 2 x 2 x 2 x 2 x 2 x 2 = 5 x 2
^{10}= 5120. - iii, stays nearly the same. The idea is that we are concerned with what the
*average*cell mass is, rather than the*total*cell mass of the culture. In exponentially growing cultures,*total*cell mass increases while*average*cell mass stays the same. - 17 x 2
^{8}/ 17 x 2^{6}= 17 x 2^{2}= 68 - iv, exponential decline
- MacConkey agar must be harsher on bacteria than is colony count agar. Consequently, though the cells are apparently all still alive in the culture being enumerated, they are declining in their health as measured by their ability to reinitiate growth on MacConkey agar. In fact, MacConkey agar is a selective medium containing bile salts, a detergent which is particularly effective against Gram positive organisms but in this case is likely affecting the older, Gram-negative bacteria being enumerated.
- 19 x 2 x 2 x 2 = 19 x 8 = 152 cells.
- 400 x 0.5 x 0.5 x 0.5 = 50 cells.
- vi, none of the above; endospore would be a correct answer.
*Bacillus anthracis*,*Bacillus subtilis*,*Clostridium botulinum*,*Clostridium perfringens*,*Clostridium tetani*.- Chromosomes are attached to the plasma membrane and consequently separate as the cell grows.
*Clostridium*or*Bacillus**3000 / 30 = 100 meaning 100 generations. This means that you will have 10 * 2*^{100}bacteria (1.27 x 10^{31}) at the end of 3000 minutes if you start with 10 bacteria. Note, if this fails to make sense, then consider that after 60 minutes you would have 10 * 2 * 2 = 10 * 2^{2}bacteria since this would have been 2 generations.- lag phase, exponential (log or logarithmic) phase, stationary phase, death phase (a.k.a., exponential or logarithmic decline).
- Cell division. [PEEK]
- microbial cell death is a state of being in which resumption of cell division, in a given environment in which the microorganism resides, is not possible, though would be possible in that environment were the cell not dead (and subsequent to the attempt to reinitiate growth is not possible after transfer to any other environment); the short answer is that the cell simply is no longer capable of dividing
- 8 generations in 10 * 60 (= 600) minutes; 600 / 8 = 75 minutes per generation
**References****Black, J.G. (1996).***Microbiology. Principles and Applications.*Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 136-140, 151-153.- Tortora, G.J., Funke, B.R., Case, C.L. (1995).
*Microbiology. An Introduction.*Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 155-158.