LAB REPORT 2

MEASURING MICROBIAL GROWTH, GROWTH

KINETICS OF MICROORAGANISMS AND
DEMONSTRATION OF EFFECT
ENVIRONMENTAL FACTORS ON BACTERIAL
GROWTH

IBG 111-INDUSTRIAL MICROBIOLOGY

PREPARED FOR:
DR. MOHD ASYRAF KASSIM

PREPARED BY: Group 6

NUM. NAME MATRIC CONTRIBUTION
NUMBER
1. DANIEL IMAN BIN SHAH 162345 OBJECTIVE,
RIDZA MATERIAL,
APPARATUS &
PROCEDURE
2. WONG KAR JIN 161937 INTRODUCTION &
CONCLUSION
3. AFIQ DANIAL BIN AHMAD 163989 RESULT &
FARID DISCUSSION
INTRODUCTION
To study microbial growth, there are several measures that can be conducted such as measurement of
optical density (absorbance) as well as biomass dry weight. These are the indicators of microbial
growth. For method of measurement of optical density, a series of 6 steps serial dilutions of the
sample given must be done. Then, the microbial growth of sample given for each step of dilution can
be imaged out by measuring the optical density of the sample after dilutions. The measurement can be
done by using spectrophotometer machine whereby OD unit will be displayed to show how
concentrated is the sample being measured. The quantification of concentration obeys the Beer-
Lambert Law whereby it states that the absorbance is proportional to the concentration of the sample.
In this experiment, we put concentrated sample to possess high microbe concentration in the sample.
Biomass dry weight is another method to quantify cell concentration in order to track microbial
growth. During the conduct of this method, centrifugation plays a crucial role in making the
quantification of cell concentration a success. Centrifugation yields supernatant and pellets whereby
pellets are the ones that determine the microbial concentration and growth while supernatant could be
the culture medium or any extracellular products produced by microbes. Decanting the supernatant
during the experiment leaving the cell and cell debris to measure the microbial growth. The pellets are
then heated by using oven-drying method of centrifuge tube with sample.

Furthermore, the growth of microorganisms can be studied by estimating the maximum specific
growth rate, μm, saturation constant, Ks and growth yield coefficient. In this experiment, there are 3
crucial components to be defined: maximum biomass concentration, maximum specific rate and
biomass productivity. In this experiment, we are given 3 different strains of microbes to determine the
components as mentioned above. Yet, components mentioned above can be detected by illustrating
graph absorbance against time that indicates their growths. The absorbance taken should be at
different timing whereby absorbance after 3 hours of incubation are taken on the experiment day and
the day after. The incubation is conducted at temperature 30°C.

Microbes have different adaptations to the ever-changing environment. There are numerous
parameters that can affect microbial growth: conditions of pH, temperature and agitation. In this
experiment, we will expose the samples given to different pH, temperature and agitation and at the
end of the experiment, we able to identify the nature of the microbe be it acidophile, alkaliphile,
neutrophile for exposure to different pH, thermophile and mesophile for exposure to different settings
of temperature as well as microbes that work well or less favorable under agitation.

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OBJECTIVE
i. To learn various methods of microbial growth estimation by counting viable cells,
measurement of optical density and measurement of the biomass dry weight
ii. To study the growth of microorganisms by estimating the maximum specific growth rate,
saturation constant and growth yield coefficient.
iii. To determine the effect environmental factors on bacterial growth.

MATERIALS

Microbes sample (saccharomyces cerevisiae), medium YEPG broth (yeast extract 1%,
peptone 2%, glucose 2%), YEPG agar (yeast extract 1%, peptone 2%, glucose 2% with
addition of agar 1.6 % w/v), saline (0.9% NaCl)

APPARATUS

Conical flasks, 250 ml, boiling water bath, analytical balance, cuvettes, conical flask (500
mL), beakers, centrifuge tubes, nutrient agar (NA), incubator shaker, spectrophotometer,
nutrient broth, test tubes, shake flasks, oven/incubator, petri dishes, desiccators, measuring
cylinder, Eppendorf tubes, bottles with diluents for serial dilution (contains 9 ml or 90 ml
Butterfields)

PROCEDURE
Part A: Measuring of microbial growth
Determination of viable cell counts
1. Serial decimal dilution were prepared and the 10−4 ,10−6,10−7,10−8 dilutions were taken for pour
plate.
2. Dilutions were incubated at 30°C for 24-48 hours.
Determination of absorbance
1. Serial dilutions of 1⁄2, 1⁄4, 1/8, 1/16, and 1/32, 1/64 were prepared by adding 50 ml sterile diluent
into 50 ml stock culture. OD was taken using a spectrophotometer at 600 nm for actual sample and
dilutions.
2. 5 samples of dilution with readings between 0.05 to 1.2 OD were taken for cell dry weight
determination.
Measurement of cell dry weight:
a. Centrifugation method
1. 1 ml of sample was pipetted into labeled Eppendorf tube respectively and was placed in
ultracentrifuge at 6000 to 10000 rpm for 5 mins. Supernatant was decanted out afterwards.
2. This step is repeated to increase the volume of culture used.
3. The pellet was washed by adding in saline equivalent to original volume of sample

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used. Pellet was vortexed and centrifuge.
4. The supernatant was decanted out and the tubes were dried in oven at 90°C for 20
Hours.
5. The tubes were removed from oven and transferred to desiccator to cooled down.
6. Tubes were reweighed.

Part B : Measuring growth kinetic of cell

1. 1 flask with 150 ml sterile broth was provided for each group. The colony bacteria were collected
from experiment 1.
2. Colony bacteria was inoculated into the culture flask and mixed carefully.
3. The culture flask was incubated in an incubator shaker at 30°C, 200 rpm.
4. Step 1 to step 6 were repeated for other two types of colony bacteria
5. 10 ml broth from the culture flask was removed and transferred to a test tube. Test tube was stored
at 0°C until required for analysis as blank.
6. 1.5mL samples from each conical flask were taken at 6pm, 9pm, 10am, 12pm and 3pm.
7. The samples were kept at 0°C until required for analysis.
8. The contents of the samples were mixed before analysis.
9. The 1.5 mL of each sample at 600 nm was read using the spectrophotometer.

Part C : Measuring effect of parameter towards growth kinetic of cell

Temperature:
1. 0.1 ml bacterial suspension pipetted aseptically into test tubes containing nutrient broth
2. Each strain was incubated at 30°C, 37°C and 40°C.
3. The rate of bacterial growth was observed on the medium after a 24 hours incubation.
4. 0.1 ml bacterial suspension was pipetted aseptically onto nutrient agar and spread the cell
over the surface of a solid agar medium with a sterile, L-shaped bent rod.
5. Incubate each strain at 30°C, 37°C and 40°C. Bacterial growth was observed.
pH:
1. 0.1 ml bacterial suspension was pipetted aseptically into test tubes containing nutrient broth of
different pH values (pH=4, pH=7, pH=9).
2. The pH tolerance of each strain was estimated by examining the growth rate of the bacteria
following incubation at 28°C for 24 hours

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3. 0.1 ml bacterial suspension was pipetted aseptically onto nutrient agar of different pH values (pH=4,
pH=7, pH=9) and the cell was spread over the surface of a solid agar medium with a sterile, L-shaped
bent rod.
4. Each strain was incubated at 37°C and bacterial growth was observed.
Agitation:
1. Pipette 0.1 ml bacterial suspension was pipetted aseptically into test tubes containing nutrient broth
(aseptically) and each strain was incubated at 50, 100 rpm and 150 rpm.
2. Observe the rate of bacterial growth on the medium after a 24 hours incubation.

RESULT

In determination of absorbance, the absorbance for each dilution was tabulated, and the
absorbance against dilution was plotted by using excel software:

Table 1: The absorbance for each dilution in determination of absorbance

Standard dilution Absorbance (at 600 nm)

1 0.080

2 0.114

3 0.150

4 0.251

5 0.434

6 0.789

Figure 1: graph absorbance against dilution in determination of absorbance

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For the determination of biomass cell dry weight, the weight for each dilution was
recorded and tabulated :

Table 2: Absorbance with cell dry weight respectively

Absorbance Weight of falcon tube (g) Net weight (g)

Dilution (at 600 nm)
Initial Final

1 0.080 13.3539 13.3543 0.0004

2 0.150 13.3739 13.3746 0.0007

3 0.114 13.3991 13.4002 0.0011

4 0.251 13.3757 13.3771 0.0014

5 0.434 13.2943 13.2961 0.0018

6 0.789 13.3123 13.3144 0.0021

The net weight obtained is then convert to cell dry weight (g / L) using the formula below:

Cell dry weight (g / L) = ( Final - Initial ) weight of falcon tube X 1000 / 24 mL

The cell dry weight was then tabulated :

Table 3: Absorbance with cell dry weight respectively

Absorbance Net weight (g) Cell dry weight (g / L)
(at 600 nm)

0.080 0.0004 0.0167

0.114 0.0007 0.0291

0.150 0.0011 0.0458

0.251 0.0014 0.0583

0.434 0.0018 0.0750

0.789 0.0021 0.0875

The results was plotted with best-fit line by using excel software which absorbance against
the cell dry weight:

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In order to determine the growth kinetic of the microbes, the microbes were cultivated in a
culture media and put in an incubator at 30 ℃, 200 rpm for 24 hours. The absorbance of the
sample was measure from t = 0 to t = 24 h, the results was tabulated :

Table 4: Optical density at different time respectively

Optical density (Absorbance measured at 600 nm)
Sample
T = 0 h T= 3 h T= 6 h T= 18 h T= 21 h T= 24 h

A 0.224 0.091 1.092 2.431 2.190 1.973

B 0.014 0.165 2.199 2.333 2.213 2.064

C 0.032 0.226 1.948 2.263 2.074 1.657

After that, by using the linear equation that had been constructed in the absorbance against
cell dry weight plot, the cell dry weight was determined:

Since the linear equation obtained was Y = 1.4786X, taking the optical density (OD) value
that was obtained in table 3, substitute the Y with the optical density and the cell dry weight
of microbes, X was calculated:

For example, sample A at T = 0 h the optical density value was 0.224, hence :

Y = Optical density
X = Cell dry weight (g / L)
Y = 1.4786X
0.224 = 1.4786X
X = 0.224 / 1.4786
X = 0.152g / L

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Hence, the cell dry weight obtained was g / L. The rest results are then using the same
method to obtain its respectively cell dry weight and tabulated as below :
Table 5 : The cell dry weight with respect to time

Cell dry weight ( g / L)

Sample
T = 0 h T= 3 h T= 6 h T= 18 h T= 21 h T= 24 h

A 0.152 0.062 0.734 1.644 1.481 1.334

B 0.095 0.112 1.487 1.578 1.497 1.396

C 0.022 0.153 1.317 1.531 1.403 1.121

The maximum biomass production, specific growth rate, maximum biomass productivity was
then obtained from the plotted graph above.

Based on graph above,

For maximum biomass production :

Maximum biomass production for sample A at T = 21 h was 1.334 g / L

Maximum biomass production for sample B at T = 21 h was 1.396 g / L

Maximum biomass production for sample C at T = 21 h was 1.121 g / L

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For specific growth rate, the formula below was used :

μ = ln (X - X0 ) / T

μ = specific growth rate

X = maximum cell dry weight

X0 = Initial cell dry weight

T = Time

For sample A,

μ = ln (1.644 - 0.734 ) / (18 - 6 )

= ln (0.91) / (12)

= 0.0786 h-1

For sample B,

μ = ln (1.578 - 1.487 ) / (18 - 6 )

= ln (0.091) / (12)

= 0.1997 h-1

For sample C,

μ = ln (1.531 - 1.317 ) / (18 - 6 )

= ln (0.214) / (12)

= 0.1285 h-1

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For maximum biomass productivity, the formula below was used:

Qm = Xm / T

Qm = maximum biomass productivity (g / L ∙ h)

Xm = maximum biomass (g / L)

T = time (h)

For sample A :

Qm = 1.481 / 21

= 0.0705 g / L ∙ h

For sample B :

Qm = 1.497 / 21

= 0.0713 g / L ∙ h

For sample C :

Qm = 1.403 / 21

= 0.0668 g / L ∙ h

For the effect of parameters (temperature, pH, agitation) towards the microbial growth
of strain C, the optical density was measured at 600 nm to determine the microbial
growth.

For temperature (30℃, 37℃, 40℃ ), the optical density was measured, tabulated and plotted
as below:

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 Table 6 : Temperature with optical density respectively

Optical density

Temperature (℃) T=0h T = 24 h

30 0.553 2.408

37 0.555 2.089

40 0.632 1.304

Figure 5: Graph optical density against temperature

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For pH (4,7,9), the optical density was measured, tabulated and plotted as below:

Table 7 : pH with optical density respectively

Optical density

pH T=0h T = 24 h

4 2.190 0.434

7 2.213 2.010

9 2.074 0.330

Figure 6: Graph optical density against pH

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For agitation (50 rpm, 100 rpm, 150rpm), the optical density was measured, tabulated and
plotted as below:

Table 8 : Agitation with optical density respectively

Agitation Optical density

T= 0h T=24h

50 0.641 1.050

100 0.509 1.112

150 0.540 1.103

Figure 7: Graph optical density against agitation

Hence, the strain B microbes was growth optimally under the condition (30℃, pH 7, 100 rpm)

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CONCLUSION

Microbial growth is a measure of the microbial progress and lividity whereby it can be determined
by several measures. The indicator that displays the microbial productivity is the quantity of biomass
produced by the microbes. In this experiment, measurement of absorbance as well as biomass dry
weight of the samples aim to track the microbial growth and productivity.

To further study the microbial growth, 3 essential components to be defined: maximum biomass
concentration, maximum specific rate and biomass productivity. In this experiment, we are given 3
different strains of microbes to determine the components as mentioned above. Yet, components
mentioned above can be detected by illustrating graph absorbance against time that indicates their
growths. The absorbance taken should be at different timing whereby absorbance after 3 hours of
incubation are taken on the experiment day and the day after. The incubation is conducted at
temperature 30°C. During the experiment, we observed that the culture solutions gradually became
cloudy which indicated the microbial growth progressing during experiment period.

In reality, microbes come across numerous obstacles whereby microbial growth can be restricted by
external and internal factors. Yet, we focused on studying the correlation between external
(environmental) factor and microbial growth. External factors such as temperature, pH and agitation
are the parameters that possibly support or hinder microbial growth which were discussed. This
experiment is a simulation of microbes in the external environment as few parameters were set to
mimic the original external environment. The nature of microbes can determined (acidophile,
alkaliphile, neutrophile for exposure to different pH, thermophile and mesophile for exposure to
different settings of temperature as well as microbes that work well or less favorable under agitation)
based on the data obtained and discussed.

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REFERENCE

1. SITNFlash. (2020, July 20). How microbes grow. Science in the News.
https://sitn.hms.harvard.edu/flash/2020/how-microbes-
grow/#:~:text=Microbial%20growth%20refers%20to%20an,made%20of%20only%20o
ne%20cell.

2. Factors affecting microbial growth: Airtek environmental: New York. Airtek

Environmental Corp. (2017, June 15). https://www.airtekenv.com/2017/06/15/factors-
affecting-microbial-
growth/#:~:text=Warmth%2C%20moisture%2C%20pH%20levels%20and,to%20the%
20life%20of%20microbes.

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