PRACTICAL 2: METHODS FOR MEASURING BACTERIAL GROWTH

1.0 INTRODUCTION

Bacterial growth is measured using two main methods, direct and indirect
methods. The direct methods actually count cells, whereas the indirect methods rely on
monitoring cell presence or activity without doing so (Libretexts, 2022). Bacterial
growth entails an increase in both size and population.

The first method is the colonies counting via serial dilution. To calculate and
observe the colony's form on the agar, the concentrated bacterial sample needs to be
diluted first until it can only produce the colony with CFUs in the range of 30-300 on a
single agar plate (Libretexts, 2022). Serial dilution is a technique that involves
repeatedly resuspending the bacteria in predetermined amounts in order to gradually
lower their concentration (“Serial Dilutions and Plating: Microbial Enumeration,” n.d.) .A
sample from each tube is plated using either the spread plate method or the pour plate
method on a solid media. Once colonies start to form, the plates are incubated. Each
dilution typically yields two to three plates, and the counts of colonies on each plate are
averaged (Libretexts, 2022).

Bacterial growth can be measured by turbidity. The growth of the bacteria is

measured through the capacity to scatter light using a spectrophotometer or
colorimeter. According to the Beer Lambert’s Law principles, the concentration of
solutes or cells is directly proportional to the absorbance (Singh, 2022). Therefore, less
light reaches the detector as there are more bacteria present in the solution. The
spectrophotometer will compute the absorbance value by comparing the detected light
to the light that was emitted. This implies that the higher the absorbance, the cloudier
the turbidity and the higher the bacterial cell count inside the nutrient broth (Singh,
2022). The advantage of the turbidity method is that it is a quick and simple method to
monitor bacterial growth (MN Editors, 2022). While the disadvantage is turbidimetry
cannot differentiate the living and dead bacteria cells as both living and dead bacterial
cells can contribute to cloudy turbidity (MN Editors, 2022).
 Measuring the microbial dry weight is the most popular method for determining
cell growth. The most accurate way to determine a cell's mass is via dry weight
technique. Cells are gathered using this technique in a liquid media. Dry weight method
consists of centrifugation which is used to separate cells growing in liquid media,
followed by the bacterial cells being cleaned, baked in an oven, and weighed
(Libretexts, 2022). The advantages of this method is that many organisms can have
their growth monitored accurately and consistently using dry weight measurements
(MN Editors, 2022). While the disadvantage is it might take a while and be delicate.
Because the size of bacteria is so tiny, it is possible to centrifuge hundreds of millilitres
to produce a suitable bacterium culture.

2.0 OBJECTIVE

To determine different methods in measuring the bacterial growth by direct and indirect
method.

3.0 REAGENTS

Serial dilution:
a) 100 mL bacterial culture
b) 6 test tubes
c) 1 mL micropipette
d) 1 mL micropipette tips (blue colour)
e) 6 nutrient agar (NA) plates
f) L-shaped glass rod
g) 100 mL beaker
h) Alcohol

Turbidity:
1. Bacterial sample (in Nutrient broth) in 100 mL Erlenmeyer flask
2. Nutrient Broth (100 mL)
3. Cuvette
4. Micropipette (1mL)
5. Micropipette tips (1mL, blue colour)
6. Spectrophotometer
4.0 PROCEDURES

Experiment 2.1 - Direct methods

Serial dilutions :

1. 6 test tubes were labelled as 10^-1 to 10^-6 with each containing 9mL of sterile
distilled water.
2. 1mL of sample was added in the first tube and the content was mixed uniformly.
3. From the first test tube, 1mL of diluted sample was taken to add to the second
test tube.
4. The same procedure was followed till the last test tube 10^-6.

Figure 1.0 shows the procedures of serial dilution.

Spread plating :

1. 0.1mL of diluted sample from test tube 10^-6 was inoculated in the appropriate
nutrient medium and was spread on plates using an L-shaped spreader.
2. The viable cells or live bacteria was multiplied and formed a colony
3. Such viable cells with the ability to form colonies are called Colony forming units
(CFU): hence, the number of cells was determined by the number of CFU/ml in
the original sample.

Figure 2.0 shows the procedures of spread plating.

Experiment 2.1 - Indirect methods

Measuring turbidity:

1. 1mL of nutrient broth was taken out and transferred into a cuvette.
2. Then, 1 mL of bacterial sample was taken out and placed into a cuvette.
3. The spectrophotometer was set at a wavelength of 600 nm.
4. The spectrophotometer was calibrated using nutrient broth.
5. The bacterial sample was placed.
6. The OD reading was observed and recorded.

Figure 3.0 shows the procedures of measuring turbidity.

Cell dry weight:

1. The filter paper weighed and the value was recorded.

2. 1 PM sample was filtered by using filter paper.
3. Then, the filter paper was left dried in the oven.
4. The dried filter paper was taken out and was weighted.
5. The value was recorded.

Figure 4.0 shows the procedures of measuring cell dry weight.

5.0 RESULT

Direct Method (Colonies counting)

a. Serial dilution using spread plating.
Formula for colony forming unit (CFU):
CFU/ml = (no of colonies x dilution factor)/ volume of culture plate (ml)

Time ( Hours ) CFU/ ml

0 0

4 6.5 x 10⁵

8 2.13 x 10⁷

12 1.4 x 10⁷

16 5 x 10⁸

20 1.5 x 10⁶

24 4.4 x 10⁷
Table 1.0 Table of Colony Forming Unit (CFU) over time

Log CFU/ml against time (Hours):

Time ( Hours ) Log CFU/ ml

0 0

4 5.8129

8 7.3617

12 7.1461

16 8.6989

20 6.1760

24 7.6434
Table 2.0 Table of Log Colony Forming Unit (CFU) over time
 Figure 5.0 shows the graph of log CFU/ml against Time (Hours)

Indirect Method
a. Turbidity
Absorbance (O.D) = 2 - log % Transmittance

Time ( Hours ) Optical density (OD) (660nm)

0 0

4 0.035

8 0.112

12 0.246

16 0.381

20 0.560

24 0.789
Table 3.0 Table of Optical Density (OD) over time
 Figure 6.0 shows the graph of Optical Density (660nm) against Time (Hour)

b. Biomass (Cell dry weight)

Time (hours) Initial weight of Final weight of Sample dry

filter paper without filter paper + weight (g)
sample (g) sample (g) (Final - Initial
weight)

0 1.0248 1.0248 0

4 1.0395 1.1200 0.0805

8 1.0495 1.1295 0.0800

12 1.0241 1.1057 0.0816

16 1.0326 1.1271 0.0945

20 1.0192 1.1190 0.0998

24 1.0181 1.1250 0.1069

Table 4.0 Table of sample dry weight (g) over time (hours)
 Figure 7.0 shows the graph of sample dry weight (g) against time (hours)

6.0 DISCUSSION

The aim of this experiment is to determine multiple methods to measure

bacterial growth, both direct and indirect. In this experiment, we used serial dilution
using the spread plate method, which is a popular direct method, as well as turbidity
and biomass (cell dry weight), which are indirect methods. The spread plate technique
in serial dilutions was used for viable plate counts, which counted the total number of
colony-forming units on a single plate (Aryal, 2022). Using a spectrophotometer,
turbidity was used to measure the cloudiness or haziness of the culture as cell
numbers increased. Microbial dry weight was calculated using biomass (cell dry
weight).

Serial dilutions with spread plating are a popular microbiology method for
determining the viable cell count or concentration of microorganisms in a given sample.
The method used began with dilutions of the sample, followed by plating samples of
the dilutions onto agar plates, spreading the mixture with an L-shaped spreader, and
allowing the microorganisms to grow into visible colonies. The initial phase in
interpreting the data is to count the colonies that have developed on the agar plates,
which are then multiplied by the dilution factor and divided by the total volume of the
culture plate to produce the estimated viable cell count per millilitre (CFU/ml). (Rijal,
2022) The standard plate count curve, or growth curve, of log CFU/ml against time in
hours was then plotted to aid in showing the correlation between them. The resulting
curve, known as the growth curve, depicts the viability and concentration of
microorganisms in the original sample. The results and patterns of the growth curve
have been analysed, and the viable cell count per millilitre increases rapidly from 0 in
the first 8 hours. It indicates that the bacteria were rapidly multiplying, and the bacterial
growth was at its highest level, which was 7.3617 CFU/ml at the hour of 8. Bacteria
divide at a constant rate throughout the log phase, and the number of bacteria grows
exponentially. (Karki, 2017) As the time passed, the viable cell count per millilitre began
to fall from 7.3617 CFU/ml to 7.1461 CFU/ml at the hour of 12. It signifies that the
bacterial growth began to stop and reduce at the slowest rate compared to the first 8
hours. The viable cell count per millilitre increases somewhat at hour 16 compared to
hour 12, increasing from 7.1461 CFU/ml to 8.6989 CFU/ml. It indicates the growth of
bacteria began to accelerate, but not as rapidly as in the first 8 hours, as some of the
bacteria died. Not all bacteria die at the same rate; some die sooner, while others are
more resistant and survive for a longer period of time. (Karki,2017) The growth graph
shows that the bacterial growth reached an exponential phase at hour 8, a stationary
phase at hour 12, and continued in the same phase for the next 12 hours until the
experiment stopped at 24 hours. There is no death phase yet because bacterial growth
requires more time than 24 hours to be monitored.

Next, the indirect method, which measures turbidity from optical density (OD).
Estimating the amount of bacteria is done through an indirect cell count. Although there
is no direct counting of bacterial cell numbers involved in this process, bacterial
colonies are calculated in order to approximate the number of bacterial cells present in
the sample (Singh, 2022). Although optical density (OD) is frequently used to assess
the density of cells in liquid culture, it is difficult to relate to actual cell count and cannot
be compared between equipment without a uniform calibration technique (Beal et al.,
2020). According to Karki (2020), total cell biomass, including both active and dead
cells, is estimated indirectly using the spectrophotometric approach. This method relies
on turbidity, where the optical density (cloudiness of a suspension) of a broth culture is
calculated to determine the bacterial population. Since turbidity is directly proportional
to the number of cells, an increase in turbidity in a culture is a sign of bacterial growth
and biomass. The graph in this experiment shows the shape of an exponential graph
where only two phases were identified, which were the lag phase and the exponential
phase. From the 0th hour (OD equal to 0) to the 8th hour (OD equal to 0.118), the
curve shows that no very significant increase was seen in the slope of the graph which
can be determined in the lag phase. Huntress (2021) stated that the lag phase
represents the time when cells are metabolically active but not dividing. Cells are
currently adjusting to and adapting to their new environment. Due to physiological
adaptation of cells to culture conditions or exoenzyme dilution as a result of initial low
cell densities, there is slow growth or no growth in the lag phase (Aryal, 2022). Since
damaged cells must first self-repair before they can begin to reproduce, there will be a
significant lag time. Normally, during the lag phase, cells are producing RNA, enzymes,
and vital metabolites that may be lacking in their new environment (such as growth
factors or macromolecules), as well as responding to environmental changes like
changes in temperature, pH, or oxygen availability (Bruslind, 2021). The next phase
was the exponential or log phase. After that, the point after the 8th hour (OD equal to
0.118) until the 24th hour (OD equal to 0.789) shows a gradually increasing slope (the
8th hour to the 12th hour to the 16th hour to the 20th hour to the 24th hour) which
points to this range according to the log phase. At this phase, cell numbers double at
regular intervals known as the mean generation time, which is a measure of the growth
rates that are optimal (Aryal, 2022). According to Bruslind (2021), predictable
population doublings occur during the exponential or log phase of growth, where 1 cell
becomes 2 cells, 4, 8, etc. When the conditions are right for the cells, they will develop
very quickly (with a steeper slope on the growth curve) but under less-than-ideal
circumstances, they will grow more slowly. Since cells in the exponential phase of
growth are the healthiest and most uniform, this explains why cells from this phase are
used in the majority of experiments. In this part, the cell growth according to the graph,
the phases that can only be seen are the lag phase and the exponential phase only.
After that, cell dry weight is the other indirect method that puts on biomass. Measuring
the microbial dry weight is the most popular method for determining cell proliferation.
Singh (2021) stated that the downside of this approach is that we might not be certain
that the dry weight is made up exclusively of bacterial cells. Additionally, it is incapable
of identifying living cells from dead cells. In this experiment, as shown by the sample
dry weight (g) against time (hours) graph, the trendline increases and has a portion of
the constant trendline and there are no trendline drops in the bacterial growth when the
time increases. From the 0th hour to the 4th hour in the graph of sample dry weight (g)
against time (hours) , the growth rate (mass of dry weight) increases, showing this
phase according to the exponential phase where the number of sample dry weight also
increases from 0 to 0.0805. According to Singh (2021), during the exponential phase,
the cells start actively dividing through binary fission, doubling in number. The cells are
currently dividing by binary fission and multiplying by two after each generation. As
DNA, RNA, cell wall components, and other components required for growth are
produced for division, metabolic activity is high. Antibiotics and disinfectants are most
effective during this growth phase because they often target bacteria's cell walls or the
processes of DNA transcription and RNA translation that are involved in protein
synthesis (Bailey, 2018). From the 4th hour to 12th hour, there were no significant
changes in the graph, as the sample dry weight did not increase or decrease too
clearly from 0.0805 to 0.0816. Then, this can be classified into the stationary phase
according to the standard bacterial growth curve. Singh (2022) stated that for a variety
of reasons, the bacterial cells begin to settle, and at this stage, bacterial development
becomes static. The metabolic activity slows down, the growth of the peptidoglycan
layer stops, and the bacterial cells adapt to survival mode during this phase, making
them less sensitive to antibiotics. It's even possible that some cells get to the
sporulation stage and begin, in theory, to produce endosperm. Then, the 12th hour to
24th hour in the graph shows an exponential phase again as there is an increase in the
curve of the graph pattern and the sample dry weight (g) increases from 0.0816 to
0.1069. The theory behind the increases in the exponential phase was just the same
as the theory behind the exponential phase before from the 0th hour to the 4th hour.
The next phase that should occur according to the standard bacterial growth curve is
the death phase, which has not happened. Huntress (2021) stated that several things
can cause an organism to enter the death (or decline) phase, such as a lack of food or
oxygen, a change in the pH of the medium brought on by cell metabolism, or a buildup
of toxic cellular waste. So, in this experiment, the death phase may not have happened
due to not having enough food, oxygen, or other factors to make the death phase
occur.

There are various possible errors in this experiment that we should be aware of,
such as improper handling of equipment and samples. Contamination may occur as a
consequence of airborne bacteria and inappropriate handling of equipment and
bacterial samples. Contamination happens when bacteria from another source are
introduced into a collected sample (Giuliano et al., 2019). Next, dilution errors, such as
errors in dilution calculations or pipetting, can have a major impact on the accuracy of
the results. Finally, poor growth control. Inconsistent control of growth conditions such
as temperature, pH, supply of nutrients, and others can cause variance in growth rates
because bacterial growth is influenced by them. Next, some precaution must be
practised during the experiment session. Firstly, when doing serial dilution, make sure
that the pipette's tip matches the volume being transferred. For instance, use a pipette
tip rated for 1 ml for transferring 1 ml of solution. Avoid touching the tubes' or wells'
edges when transferring solutions to reduce the possibility of cross-contamination
(Jeetendra, 2023). Other than that, maintain the spectrophotometer clean. Even the
most precisely calibrated instrument won't provide accurate or reliable data if it isn't
clean. Since dirt, dust, grime, and other impurities can skew measurement results, it's
crucial to undertake regular cleanings to keep the spectrophotometer clean (Phillips,
2022). Last but not least, according to Hill (2019), carefully handle the weights. Avoid
touching the weights with bare hands as this can lead to inaccurate readings. Always
place the samples using a pair of clean forceps. Gently place the samples in the pan's
centre. Don't leave the weights outside the workstation after you've finished using
them.
7.0 CONCLUSION

To conclude, the aim of this experiment is to determine different methods in

measuring the bacterial growth by direct and indirect method. Serial dilution was
performed by using the spread plate method as a direct method while turbidity and
biomass cell dry weight is an indirect method. The bacterial growth can be calculated
by using cell dry weight methods. Both direct and indirect techniques are successfully
performed to measure the bacterial growth. The bacterial colonies have been observed
and it is increased in the turbidity and biomass method which indicate the bacterial
growth.

8.0 REFERENCE

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