COLLEGE OF AGRICULTURAL AND ENVRONMENTAL SCIENCES

SCHOOL OF FOOD TECHNOLOGY, NUTRITION AND BIO-

PROCESSING ENGINEERING
DEPARTMENT OF FOOD TECHNOLOGY AND NUTRITION

NAME: ATUKUNDA ROSETTE

REG.NO. 23/U/06920/PS

STUDENT NO. 2300706920

COURSE CODE: FST2103

COURSE NAME: FOOD MICROBIOLOGY II

Title: MICROBIAL GROWTH CURVES

Introduction
Microbial growth has been recorded the most common cause of food spoilage and of food
poisoning when a pathogen is involved. Microbial growth has been described using several
models, some of which were developed in the context of food quality and safety and
primarily describe and predict microbial growth in what is approximately a “closed habitat”
where all life-sustaining resources are finite and non-renewable, there is no input or output of
any more nutrients during the incubation and the metabolites excreted by the living cells, and
the cells that have died are not removed. Thereby, the amount of nutrients decreases while the
rate of metabolic wastes increases. This is usually also referred to as the batch culturing.
Rather the case with the continuous culturing, for example fermentation process where
resources are continuously replenished to maintain the same concentration of nutrients and
metabolites or cells are harvested. Microbial growth in yeast cells occurs principally at
optimal conditions of culture media and physiochemical parameters such as temperature, PH,
salinity, available nutrients through cell division during asexual reproduction either by
budding where a daughter cell grows from the parent cell as an outgrowth or fission where
the parent ell divides into two new daughter cells resulting into an increase in the number of
the individual cells though at a times the growth can be used to mean increase in the size of
the cell. The increase in the number of individual cells can be measured as a function of time
to obtain a growth curve represented by the logarithm of the number of cells as a function of
time with organisms and can adopt a wide range of rates from zero growth to the maximum
achievable growth (Peleg et al, 2011)

The main objective of the practical was to determine the growth of yeast cells present in a
sample of banana juice and illustrate the growth using the standard microbial curve.

The typical growth curve of micro-organisms in a closed habitat has four discernible stages
usually referred to as phases, that is the lag phase, log or exponential growth phase, stationary
phase, and the death or mortality phase. However, most of the models presented and
discussed in the food microbiology literature, usually deal with only the first three since most
foods become unsafe for consumption long before massive microbial cell death begins or
even before the stationary phase has been reached. The shape of the microbial growth curve
is sigmoid and reflects events at cellular level regulated by biophysical and biochemical
processes involving anabolic and catabolic reactions inside and outside the cells. The actual
curve records the countable cells determined at certain time intervals during the population's
evolution and were commonly expressed in the form of Colony Forming Units, CFU's
excluding the injured cells or any other that failed to form colonies (Forget et al, 2010).
Methodology
Materials used
 Acidified potato dextrose (PDA) o Incubator set at 370C
agar, selective media for yeasts
o Fresh banana juice
 Sterile Ringer’s solution
 Sterile pipettes, test tubes and petri o Cotton wool
dishes
o Permanent marker
 Pipette pumps
 Water bath o Camera
 70% ethanol
o Notebook and pen
Procedure
The glassware like pipettes wrapped in aluminium foil and petri dishes were sterilized in the
hot air oven at 1800C for 30minutes
Flames were set up on the Bunsen burner to provide a sterile working environment to avoid
contamination of the samples and apparatus
The table surfaces and hands were disinfected with 70% ethanol to kill the present micro-
organisms avoiding any contaminations of our samples and media during the experiment.
The fresh banana fruit juice was aseptically divided into 8 test tubes and stored for
0,2,4,6,12,24,36,48 and 60 hours respectively.
After the appropriate time for the sample in each test tube was serially diluted, three dilution
factors selected and pour plated.
The petri dishes were well labelled with the dilution plated and the name of the group using a
permanent marker.
3 selected dilution factors were selected from each portion and then 1ml was plated on sterile
petri dishes in duplicates using sterile pipettes and a pipette pump.
The already prepared tampered acidified Potato Dextrose agar sterilized in the autoclave at
121 0 C was then poured on the petri dishes.
The petri dishes were then gently swirled to mix the media and the sample well and left to
solidify.
The petri dishes were then incubated in an inverted position at 37 0C for 72 hours to allow for
microbial growth.
The washable glass ware such as pipettes were washed thoroughly with detergent, rinsed with clean
water and the work surfaces cleaned.
The number of colonies were then counted, recorded and the microbial concentration
calculated in colony forming units per ml using the formula below to determine the survival of micro-
organisms at different lactic acid concentrations.

C=
∑X
V ¿¿
Where N is microbial count, x is the number of colonies, V is the volume of sample
plated, N1 is the number of plates at 1st counting, N2 is the number of plates at second counting
and d1 is the dilution factor plated.
The log microbial concentration was calculated and a microbial growth curve plotted.
Results
Time Microbial concentration Log (Microbial
(Hours (CFU/ml) concentration)
)
O 1.76×105 5.2455
2 1.29×105 5.1105
4 2.24×105 5.3502
6 3.89×105 5.5899
12 1.5×108 8.1761
24 1.14×109 9.0569
36 4.435×109 9.6469
48 2.4×107 7.3802
60 1.47×108 8.1673

Microbial curve for yeasts in banana juice over

60 hours.
12
log (Microbial concentration)

10

0
0 10 20 30 40 50 60 70
Time in hours

Discussion
The lag phase was illustrated on the growth curve from a period of 0 to 6 hours during which
there was practically slow growth, characterized by little or no cell reproduction , synthesis of
cellular components for example initiation of the metabolic machinery for growth like
replication of the genetic material and basal metabolism and intracellular changes in an effort
to adapt to the new environment after colonization; it was also referred to as a phase when the
population initially declines or a stationary phase develops before the exponential phase can
be clearly identified Its duration is usually dependent on the availability of nutrients,
moisture, PH and the environment (Yates et al, 2007)
The log or exponential phase was seen in the period from 6 to 12 hours during which the
increase in cell abundance was exponential. During this phase, cells reproduced at a rate
proportional to the number of cells leading to an exponential increase in the number of
cellsand the generation time which is the number of days it takes to double the number of
cells in the culture and is calculated as the natural logarithm of 2 divided by the growth rate
oughts to be constant; During this stage micro-organisms are very destructive that is are
capable of producing slimes, toxins, off odors and colors in food which are associated with
spoilage and food borne illness. At the inflection point of the exponential phase of growth,
cells showed growth to have been at its maximum (optimum) growth and there was
significant competition for the available nutrients, which later leads to a progressive decrease
in their growth rate to a gradual growth rate between the 12 and 24 hours.
From 24 to 36 hours, the stationary phase was illustrated by a period of slow growth though
is usually represented by zero net growth in the typical growth curve. During this phase, the
cell numbers did not change due to the equilibrium between cellular division at a limited rate
by the cells that can take advantage of the released nutrients as a result of cell lysis and death
of some cells induced by nutrient depletion due to the competition, accumulation of wastes or
any adverse environmental conditions that may kill the yeast cell;
Micro-organisms at the stationary phase or the steady state (zero) growth are undergoing a
stage of adapting from growing to maintenance or adverse conditions so that they can persist
as long as possible; as they maintain cellular machinery, use their scarce excess energy with
minimum losses and synthesis of organic matter to increase their biomass at the rate allowed
by the available nutrients and the surrounding conditions. This is a character exhibited by the
persistence of viable and active cells of soil thermophiles and other bacteria like Lactococcus
lactis, Bacillus subtilis, Pseudomonas putida and yeasts like Saccharomyces cerevisiae.
During this phase microbes develop different strategies of survival for example activating the
machinery to generate metabolites such as antimicrobial substances, vitamins or toxins that
are not needed for growth can be beneficial in the times of adversity to outcompete other
species or provide protection in times of starvation. The gram-positive bacteria and fungi
have developed morphological differential structures known as spores, with strong cellular
envelopes to protect the genome, minimum machinery to restart growth once conditions get
favorable in order to persist in the challenging conditions. Others without resistance
structures persist through morphological changes like changing cell length increase due to
inhibition of cell division or restructuring the whole cell genome to repress common
metabolic processes.
Stationary phase promoters that orient cells for survival with interesting potential for
biotechnology and large-scale protein production also exist, with the increasing levels of
toxic by-products, the cells reprogram their cellular machinery for adaptation for the new
conditions due to the unique components such as the CASP (Constant Activity at Stationary
phase) important in protein synthesis, activation of ppGpp and pppGpp which diverts the
cells to consuming their own biomass resources such as reserves and amino acids to extend
their survival time and activate maintenance processes, the alternative sigma factor Rpos that
play the decisive role, the production of GASP ( Growth Advantage in Stationary Phase)
phenotype mechanism due to mutations of the Rpos gene for an advantage in competing the
parental cells at prolonged starvation by scavenging the nutrients from the lysed cells,
common in prokaryotes and the also the SCDI (Stationary phase Contact-Dependent
Inhibition) phenomenon that inhibit growth of contacting cells to avoid competition.
As the stationary phase of growth progressed, conditions became increasingly adverse and so
growth became more limited over time. Cell death increased, thus a reduction in numbers that
resulted in an asymptotic shape in the final portion of the growth curve leading to the decline
or decay (death) phase of growth at the advanced stages, the decay phase was aimed at
reducing the number of cells in the culture so that some could survive and the achieve a much
longer survival time at a cost of losing a large fraction of its members (Gonzalez et al, 2023).
The death phase was noticed from 36 to 60 hours. This was a period presenting negative
growth that is decay of cell number or biomass or typically no growth due to a variety of
factors such as antimicrobials, starvation due to nutrient exhaustion, adverse conditions like
increased toxicity, change in pH. and temperature.
There was an abnormal increase in the concentration of yeast cells after the 48 hours which
could have been due to contamination during the culturing process.
Conclusion
Therefore, there is a limit for the potential uses of microorganisms and the appropriate
management of ecosystems due to lack of knowledge about the microbial world.

Manipulation of the different phases of the growth curve for example

prolonging the lag therefore delaying the onset of the log phase can be
used in preserving foods to achieve longer shelf-lives and applied in such
processes as safe food production, wastewater treatment, bioremediation,
or microbe-mediated mining (Esser et al, 2015).

References
1. Peleg, M., & Corradini, M. G. (2011). Microbial growth curves: what the models tell
us and what they cannot. Critical reviews in food science and nutrition, 51(10), 917-
945.
2. Forget, N., Belzile, C., Rioux, P., & Nozais, C. (2010). Teaching the microbial growth
curve concept using microalgal cultures and flow cytometry. Journal of biological
education, 44(4), 185-189.
3. Esser, D. S., Leveau, J. H., & Meyer, K. M. (2015). Modeling microbial growth and
dynamics. Applied microbiology and biotechnology, 99, 8831-8846.
4. Gonzalez, J. M., & Aranda, B. (2023). Microbial growth under limiting conditions-
future perspectives. Microorganisms, 11(7), 1641.
5. Yates, G. T., & Smotzer, T. (2007). On the lag phase and initial decline of microbial

growth curves. Journal of Theoretical Biology, 244(3), 511-517 .