Group #4

Group Members: Date Performed: July 3, 2019

Rejuso, Alyssa Rae Date Submitted: July 8, 2019
Romero, Timothy
Ruiz, Kim
Sacro, Rico Rafael
Sanchez, Kristel
Santiago, Carl Vincent

Exercise No. 6
Bacterial Growth Curve

Abstract

Determining the microbial cycle is vital in understanding the overall concept of growth of
microorganisms. This can primarily be presented by the four stages in a bacterial growth curve (BGC). Lag
phase is where primarily replication of cellular components occur. The logarithmic or exponential phase
exhibits growth in cell density. Stationary phase shows decreased net growth of bacterial cells. This
transitions to death phase, where net loss of cells is due to lack of nutrients and overwhelming amount of
toxic wastes. In order to propose an ideal BGC, unknown bacteria was incubated and observed over the
course of 48 hours. A BGC was formulated from this scrutinization. Findings are as follows: lag, log,
stationary, and death phases lasted 8, 16, 12, and 12 hours respectively. Cells during the lag phase
indicated an increase in cellular material rather cell growth, due to important acclimation to agar culture
environment. The stationary phase indicates rate of cell growth equal to rate of cell death due to limited
nutrients and accumulating waste. The death phase indicates rate of cell death outcompeting the rate of
replication, caused by increased metabolic waste and little to no nutrients available for cell growth. As
opposed to batch cultures, chemostat is an open continuous culture that maintains bacteria at log phase
through influent of new medium and effluent of old medium, usually carrying waste and dead cells.

Keywords: bacterial growth curve, microbial cycle,

I. Introduction
By general knowledge, bacterial growth is referred to as the increase in cell number rather
than cell size. These cells undergo cell division, either through binary fission or budding, in order
to manipulate proliferation. Their growth, generally, is deemed to be exponential--where the
number of cells doubles in a constant time interval. However, this is only a part of the overall
microbial cycle (Madigan et al. 2019).
Information about microbial growth is often obtained from controlled laboratory
experiments using pure cultures of certain microorganisms. With this, there are two common
approaches in order to further study the growth and cycle of microorganisms: closed culture
system (batch culture), and open culture system (chemostat).
A batch culture aids in the growth of organisms, however, nutrients will eventually be
scarce after a certain period of time. While in a chemostat, nutrients are continuously provided
to the microorganisms, and metabolic wastes are filtered out via the overflow. However, in order
to study the growth curve of an organism, an enclosed vessel or batch culture is used.
A growth curve is a representation of the complete microbial cycle of a microbe, and is
comprised of four stages: lag, logarithmic (or exponential), stationary, and death (Maier et al.
2009).
 The lag phase is commonly the first stage of a bacterial growth curve (BGC). The growth
rate during this period is essentially zero; noting that cells only increase in size (e.g. replication of
DNA) rather than executing cell division to increase cell density. Given that an inoculum is
transferred to a different medium, bacterial cells are adapting to the new environmental
conditions they have been introduced to. Hence, the lag phase is also referred to as the
acclimation stage. Afterwhich, where cells begin to double its initial cell count, microbial cycle
will transition to the logarithmic stage.
The logarithmic phase is the period where cell density continues to double, given that the
nutrients in the medium are still available. During this stage, the microorganisms tend to exhibit
exponential growth-- the most rapid growth possible in a microbial cycle given the conditions a
batch culture presents. In relation to this, the generation time is primarily determined. Generation
time is the amount of time it takes to double the initial population of the bacteria. However, this
immensely varies on growth conditions and genetic characteristics of the organism itself (Madigan
et al. 2019).
The stationary phase, on the other hand, is the period where the rate of cell production
equals the rate of cell death, thus no net cell growth happens. This is often due to the declining
amount of nutrients present in the medium. Given this, the environment starts to exhibit toxic
and detrimental conditions-- where a massive decrease in pH and oxygen levels occur. After this
stage, cells will slowly transition to the final period of the growth cycle: death phase.
The death or decline phase is often characterized by the net loss of viable and culturable
cells. Similar to the logarithmic phase, this also occurs at an exponential rate. Due to the
overwhelming amount of toxins and wastes present in the environment, these cells start undergo
involution (cell-shrinking). In addition, for genera that produce endospores, the number of these
endospores will eventually surpass the amount of vegetative cells. Thus, concluding the microbial
cycle (Maier et al. 2009).
Population growth is relevant to ecology, and significant in examining various metabolic
activities In order to understand the concept of growth for microorganisms, microbial cycle must
be studied. Hence, this research has the following objectives: a.) to characterize the different
phases of the bacterial growth curve, and b.) construct a bacterial growth curve (ADMU n.d.).

II. Materials and Methods

Bacterial Growth Curve. The values used in order to graph the bacterial growth curve was
obtained by monitoring and observing the growth of a bacterial culture for 48 hours at a
temperature of 37oC in a closed culture system (batch culture). The three replicates of the
population count (CFU/mL) was then averaged and converted to its logarithmic values. By using
Microsoft Excel, the points were then graphed where y is the logarithm of the average population
count in CFU/mL, and x is the incubation time in hours. The different phases of the bacterial
growth curve was also identified (ADMU n.d).

III. Results and Discussion

Ideal Bacterial Growth Curve Phase for the Observation of Physiological and Biochemical
Characteristics of a Bacterial Cell. The physiological and biochemical characteristics that are
commonly used to identify bacteria are manifested during the logarithmic phase of bacterial
growth. At this phase, bacterial population is nearly uniform in terms of their metabolic activities,
chemical cell composition, and other physiological mechanisms. Contextually, all bacteria
undergo rapid stage of cell division and exhibit balanced growth during this phase.
Correspondingly, this is also where generation time of the bacteria can be procured since bacteria
divides continuously at a constant rate and the bacterial number proliferate exponentially.
Moreover, this phase takes place for several hours depending on the type of organism, density of
organism, and conditions of growth (Karki, 2017). Taking the preceding notions into
consideration, another scientific corroboration denotes that physiological and biochemical
characteristics of a bacterial cell must be studied during the lag phase, or the phase of
acclimatization and adaptation to new environment, since bacteria might have different
physiological growth phases and each species has its unique growth rate; cells require time and
a certain cell size and mass to initiate cell division (Singh 2018). This study also denotes that
microbial lag phase is a very crucial stage of bacterial growth since it allows one to understand
the physiological and regulatory process responsible for adapting or acclimatizing to new
environment. Lag phase allows the adaptation required for bacterial cells to new environmental
conditions and this stage constitutes the repair of bio-molecular damage and the synthesis of
cellular components mandatory for growth. This also manifests that cells are metabolically active
but not growing in number during the lag phase, however, it also questions the notion if they are
just repairing and synthesizing bio molecules (Singh 2018).

Table 1. Population count (CFU/mL) of a bacterium over 48 hours.

Incubation Population Count (CFU/mL)
time (h)
Replicate 1 Replicate 2 Replicate 3 Average log

0 1.0 x 102 1.1 x 102 1.2 x 102 1.1 x 102 2.0

4 1.3 x 102 1.0 x 102 1.0 x 102 1.1 x 102 2.0

8 1.0 x 102 1.3 x 102 1.0 x 102 1.1 x 102 2.0

12 1.5 x 104 1.5 x 104 1.5 x 104 1.5 x 104 4.2

16 1.6 x 105 1.4 x 105 1.5 x 105 1.5 x 105 5.2

20 2.0 x 106 2.1 x 106 1.9 x 106 2.0 x 106 6.3

24 1.0 x 107 1.2 x 107 1.0 x 107 1.1 x 107 7.0

28 1.0 x 107 1.0 x 107 1.0 x 107 1.0 x 107 7.0

31 1.0 x 107 1.0 x 107 1.0 x 107 1.0 x 107 7.0

36 1.0 x 107 1.0 x 107 1.0 x 107 1.0 x 107 7.0

40 3.0 x 106 3.0 x 106 3.0 x 106 3.0 x 106 6.5

44 1.0 x 105 1.0 x 105 1.0 x 105 1.0 x 105 5.0

48 3.0 x 104 3.0 x 104 1.0 x 104 2.3 x 104 4.4

 Figure 1. Graph of a bacterial growth curve of a bacterium over 48 hours
The graph presented the following: lag phase occurred until the 8th hour. Logarithmic
phase lasted for 16 hours (8th to 24th hour). The stationary phase transpired and lasted for 12
hours (24th to 36th hour). Death phase gradually occurred over the course of 12 hours (36th to
48th hour).

Lag phase of Bacterial Growth Curve. The lag phase of the bacteria lasted for 8 hours after
incubation. Immediately after inoculation of cells into a fresh medium, a microbial culture’s
population remains temporarily unchanged, and growth begins only after a period of time called
this phase. Here, cells may be growing in mass or volume, as well as synthesizing enzymes and
proteins, and increasing in metabolic activity. The lag phase is theoretically always observed,
however, the length of this phase is dependent on an array of factors. These include the size of
the inoculum, the necessary recovery time from physical damage in the transfer, the time
required for synthesis of essential enzymes, and time required for synthesis of inducible enzymes
necessary for metabolism. If a culture that is exponentially growing is moved into a medium of
the same composition and growing conditions, there will be essentially little to no lag, and log
growth will begin immediately. If the inoculum is taken from an old culture, there is usually a lag,
as the cells are depleted of many essential components, and time is required for their biosynthesis
(Madigan et. al 2019).

Importance of the Stationary Phase. The stationary phase is often instigated by a growth-limiting
factor such as essential nutrient depletion, and/or inhibitory product formation such as an organic
acid. Stationary phase arises from a situation in which growth rate and death rate of cells are
equal. Whereas, the number of new cells created is limited by the growth factor. As a
repercussion, the rate of cell growth matches the rate of cell death. Thus, the graphical
representation of the stationary phase is a smooth, “plateau-like”, or horizontal linear part of the
bacterial growth curve. Hereafter, the importance of the stationary phase is to detect mutations.
 As such, there is an evidence that DNA damage is accountable for many of the mutations arising
in the genomes of stationary phase or starving bacteria. Endogenously generated reactive oxygen
species are corroborated to be a major source of such damages (Bridges et al. 2011). Furthermore,
this phase also manifests if there is a prevailing level of damaging pH, depleting level of oxygen,
or diminishing level of nutrients (Madigan et al. 2019).

Causes of the Decline Phase. In the death or decline phase, the number of viable cells decreases
exponentially, as well as the population growth. This is caused by the reduction in the availability
of nutrients, and the increase in the number of waste products present. The temperature may
play a role in the depletion in the number of living cells as it may be above or below the threshold
of the species present (Bailey 2018). When cells from this phase were then transferred into a new
and fresh culture medium, they were unable to grow. Thus, many scientists believe that the
conditions in the culture may have been detrimental for the cells present that the cells were
irreparably harmed (Bruslind n.d).

Logarithmic Phase in Continuous culture. The second stage in the bacterial growth curve is the
logarithmic phase, where bacteria have acclimated to the controlled environment and are capable
of doubling in numbers (Madigan 2019). However, bacterial growth differ in closed systems, like
the conventional batch system, as opposed to open systems, like continuous culture. The
chemostat is a continuous culture that maintains a fresh supply of bacteria through the renewal
of nutrients by continuously adding an influent of fresh medium, which is followed by the removal
of waste by disposing spent medium as effluent (Madigan 2019; Maier and Pepper 2015). The
environment simulated within batch cultures changes over time due to the increase of bacterial
cells given a set amount of nutrients only made available to them, which gives rise to the four
phases of the bacterial growth phase; lag, log, stationary, and death phases. (Doran 2013;
Madigan 2019). Meanwhile, chemostat overhauls these steps by achieving a steady-state, where
population has an equal rate of bacterial replication to the rate of removed bacteria (Allen and
Waclaw 2018). This equilibrium allows a controlled population in cultivating bacteria, specifically
suspending bacteria from moving past the logarithmic phase in bacterial growth curve. The
available bacteria in the population will remain in logarithmic phase, which is favorable especially
for keeping a healthy source culture (Madigan 2019).

IV. Conclusion and Recommendation

The researchers were able to construct a bacterial growth curve using the data provided
by the laboratory instructors. This bacterial growth curve was plotted using the values of time
(hours) in the x-axis and the population count (CFU/mL) in the y-axis. After analyzing the graph,
each phase of the bacterial growth curve was determined based on the trend of the values. Every
phase was discussed by describing the physiological and biochemical properties of the bacterial
cells in each phase as well as the conditions of the environment.
The four phases of the bacterial growth curve include, lag phase, logarithmic phase,
stationary phase, and death phase. Lag phase happens immediately after inoculation from a
media into a new media with different components. In this phase, the bacteria are adapting to its
environment before they undergo logarithmic phase. In logarithmic phase, bacteria will start to
replicate exponentially and the generation time of the bacteria can be determined. After a certain
period of time, the amount of nutrients would diminish resulting in an equal rate of cell
production and cell death. This would continue until the number of viable cells starts to decrease
 exponentially due to insufficient nutrients and the increased presence of waste materials. This
phase is referred to as death phase
All of these phases occur in a closed culture system while in an open culture system such
as a chemostat, there are no stationary and death phases. This is due to the constant supply of
nutrients supplied in the chemostat resulting into a continuous logarithmic phase.
In the graph of the growth curve of an unspecified bacterium, the lag phase lasted for 8
hours, the logarithmic phase occurred the longest at 16 hours, the stationary phase stayed for 12
hours, and the death phase followed for 12 hours.

V. References
Allan R, Waclaw B. 2019. Bacterial growth: a statistical physicist’s guide. Reports on Progress in
Physics. Physical Society (Great Britain). 82(1)

Ateneo de Manila University. [date unknown]. Introduction to microbiology laboratory manual.

Bailey R. 2018. Phases of the bacterial growth curve. [Internet]. ThoughtCo; [cited 2019 Jul 7].
Available from https://www.thoughtco.com/bacterial-growth-curve-phases-4172692

Bridges BA, Foster PL, Timms AR. 2011. Effect of endogenous carotenoids on adaptive mutation
in Escherichia coli FC40. Mutat Res [Internet]. [cited 2019 Jul 7]; 473 (1): 109-119.
Available from: doi:10.1016/s0027-5107(00)00144-5

Bruslind L. [date unknown]. Microbial growth. [Internet]. Oregon State University; [cited 2019 Jul
7]. Available from http://library.open.oregonstate.edu/microbiology/chapter/microbial-
growth/

Karki G. 2017. Bacterial growth curve [Internet]. Bhaktapur (NP): Online Biology Notes; [cited
2019 Jul 7]. Available from onlinebiologynotes.com/bacterial-growth-curve

Madigan M, Bender K, Buckley D, Sattley W, Stahl D. 2019. Brock biology of microorganisms.

15th Edition. Harlow (UK): Pearson Education Limited. 1064 p.

Maier R, Pepper I, Gerba C . 2009. Environmental Microbiology. 2nd Edition. Amsterdam (NL):
Elsevier. 598 p; [cited 2019 Jul 7]. Available from
booksite.elsevier.com/samplechapters/9780123705198/Sample_Chapters/04~Chapter_
3.pdf

Maier R, Pepper I. 2015. Bacterial growth. Environmental Microbiology. Third Edition. Academic
Press: p. 37-56.

Doran P. 2013. Homogeneous reactions. Bioprocess Engineering Principles. Second Edition.

Elsevier: p. 599 - 703
Singh A. 2018. Microbial lag phase [Internet]. Berlin (DEU): ResearchGate; [cited 2019 Jul 7].
Available from researchgate.net/topic/Bacterial-Growth-Curve