UNIVERSITY OF SCIENCE - VNU HCM

FACULTY OF BIOLOGY & BIOTECHNOLOGY

REPORT OF LABORATORY
MICROBIOLOGY

Lecturer Ph. D NGUYEN THI MY TRINH

MSc. NGO THI HUYEN TRANG

GROUP 1: NGUYEN HONG AN – 20157068

LE MINH TUAN – 20187215
NGUYEN THI ANH TUYET – 20187218

Ho Chi Minh City, April 2024

 I. Microorganism observation
❖ Colony observation
1. Principle:
Based on the characteristics of the original bacteria, we can choose the appropriate
media, culture method, and incubation time. As in this experiment, we diluted and
cultured sample S1 on LB and PGA media. After carefully observing the microbial
colonies on the agar surface, we can examine the colony morphology, such as size,
shape, color, texture, height, and any other characteristics. any other distinction.
2. Protocol:
 3. Result:
LB plate: colonies are off-white with small white dots as droplets of water, the
plate is wet (bacteria is growing).
PGA plate: colonies are larger, dark green or gray, fibrous and the plate is dry
(fungi is growing).
4. Conclusion:
Various culture media are tailored to specific types of microorganisms, with some
media exclusively supporting the growth of specific species or favoring their
dominance (e.g. LB medium supports the growth of bacteria, and PGA medium
supports the growth of mold...). The number of microorganisms per plate also varies
depending on the dilution level; For example, an LB plate diluted 10 -3 will have less
growth than a plate diluted 10-2.
 Microscopic observation
1. Principle
Steps such as sample preparation, choosing the right microscope, adjusting
magnification and focus, and using appropriate lighting will allow us to analyze
microbial morphology and internal structure. Assess characteristics such as cell shape,
size, arrangement, presence of flagella or pili, and the presence or absence of specific
organelles or inclusions. From there, microorganisms can be identified and classified.
 2. Protocol

3. Result
Bacteria: Unicellular, small, rod-shaped
Yeast: Unicellular, shoots can be seen growing
Fungi (mold): filamentous, with sporangia
Actinomyces: microfiber, big cluster
4. Conclusion:
In summary, microscopic observation of microorganisms provides valuable
insights into the morphological characteristics and internal structures of
microorganisms such as pili, flagella, cell arrangement, or size. Through this
experiment, we gained a deeper understanding of their current morphological
characteristics.
❖ Gram staining:
1. Principle
The principle of the Gram staining method is based on the ability of bacteria to
retain or lose their crystal violet color during the staining process. The process
includes steps of staining with crystal violet solution, iodine solution, decolorization
with 95% alcohol solution, counterstain, and observation. Gram-positive bacteria
retain their purple color, while Gram-negative bacteria lose their purple color and are
stained red or pink. This is an important method for classifying and identifying
bacteria in microbiology based on differences in their cell wall composition.
2. Protocol
3. Result
 Looking at the gram stain image with a microscope, it can be seen that both the
control sample and the U1 sample appear purple and pink-red, meaning that these two
samples contain both gram-positive and gram-negative bacteria.
 According to the correct procedure, sample U1 can only have one type of
bacteria, but here both types appear, meaning that during the operation we may have
accidentally cross-contaminated the two samples. We do not know exactly whether
the U1 sample that our team tested is a gram-positive or gram-negative bacteria, but
based on the figure, we can see that the pink bacteria group is more present than the
purple one, so our team predicts the U1 sample may be gram-negative bacteria.
4. Conclusion
In conclusion, the Gram staining process is an essential technique used in
microbiology for the differentiation and classification of bacteria based on their cell
wall characteristics. The Gram staining procedure successfully revealed the
differential staining characteristics of the bacterial cells. The observed results indicate
the presence of both Gram-positive and Gram-negative bacteria within the sample.
These findings offer crucial insights into the composition and diversity of the
microbial population under study.
II. Isolation lactic acid bacteria:
1. Principle
Based on sampling yogurt containing these bacteria, then inoculating it into an
MRS-CaCO3 selective medium using the 4-way-streaking method will promote the
growth of lactic acid bacteria while inhibiting other microorganisms. other object.
After that, the plates are incubated under appropriate conditions to isolate lactic
bacteria based on morphological characteristics, size, and color... From there, a
characteristic colony is captured and transferred to the LB medium for observation to
viewing.
2. Protocol

3. Result and discussion:

Observe the results showing a reaction between lactic acid and CaCO 3, indicated
by a faint white halo surrounding the colony. Using the 4 way-streaking method, in
the later parts these colonies become fewer, gradually sparser, and single colonies
appear.
When capturing a typical colony on the MRS-CaCO 3 plate to transfer to the LB
tube, you can see the solution becoming cloudy and the phenomenon of suspension in
the test tube.
4. Conclusion:
The MRS-CaCO3 plate makes it very simple to identify the growth of lactic acid
bacteria since the CaCO3 diffuses around the colonies in a white hue.
III. Microbial quantification
1. Principle:
The principle of microbial quantification involves determining the number or
density of microorganisms in a sample. This is usually done by culturing the sample in
a growth medium, allowing the microorganisms to grow under appropriate conditions,
and then counting or estimating their numbers. In this experiment, we used E. coli as a
sample, diluted it to a concentration of 10-7, and then plated it. After incubation for
enough time, we count and calculate the number of microorganisms. Combined with
the use of a spectrometer at a wavelength of 600nm to determine the optical density,
from there we can build a linear correlation graph.
2. Protocol:

3. Result:
N (number of cells per milliliter) of each sample used for producing the Standard
Curve is selected by comparing the number of colonies on the plate after counting the
number of colonies on three different dilutions of each E. coli sample that is provided.
Calculate the number of colonies per ml (cfu/ml) of each culture
● Number of cells per ml (cfu/ml) = number of colonies x dilution factor x 10
(cfu/ml)

Level dilution 10-5 10-6 10-7

A (total colonies) 154 50 103
 N (CFU/ml) 1.54 x 108 5 x 108 1.03 x 1010
 Looking at the data we collected, we can also see that this number is not
accurate compared to the standard. The higher the dilution concentration, the
number of colonies must decrease according to the dilution level, but our numbers
are quite precarious. This may be because our operation had errors during
implementation.
The result of group A (groups 1 through 5) following the counting and selection of
colonies of the sample.

Group OD Log(N/ml)
1 0.119 9.56
2 0.262 9.08
3 0.307 9.77
4 0.431 9.95
5 0.543 9.94
  Looking at the graph, it's evident that our standard curve is quite skewed,
with a confidence level of only 39%, a figure too low for us to rely on. Therefore, our
team has decided to replace this figure. At our group's concentration of 0.1, we'll
substitute the data from Group 1 to Group 13 to generate a new linear correlation
graph.

Group OD Log(N/ml)
13 0.12 8.67
2 0.262 9.08
3 0.307 9.77
4 0.431 9.95
5 0.543 9.94
 The result of ΔOD 600nm of sample Q is 0.321, dilution = 3.

4. Conclusion:
With the original data (OD600nm=0.119, Log(N/ml) = 9.56) and the substituted
data (OD600nm=0.12, Log(N/ml) = 8.67), the results obtained for sample Q were 1.32
x 1010 CFU/ml and 8.34 x 109 CFU/ml, respectively. However, with the low
confidence R values for both equations (both 0.39 and 0.8 are smaller than 0.96), our
team has concluded that the quantification results are unreliable.
IV. Enzyme activity
❖ Catalase test:
1. Principle
The catalase test is based on the detection of the catalase enzyme in
microorganisms. Catalase helps break down hydrogen peroxide into water and
oxygen. The test involves cultivating 3 bacterial samples in LB agar medium, then
dropping hydrogen peroxide into the cultured samples and observing the occurrence
of air bubbles indicating the presence of catalase.
2. Protocol
 3. Result
The catalase test was carried out by touching an anse ring incubated in 24 hours
LAB strain into a few drops of H2O2 on a Petri. If no bubbles appear, the organism is
catalase-negative, and vice versa. The formation of bubbles indicated the positive
reaction on the isolate at Bacillus subtilis, which is the formation of oxygen due to the
breakdown of H2O2 by the catalase enzyme. Lactobacillus and Escherichia Coli are
negative for starch non-hydrolyzing.
4. Conclusion:
The catalase test is used to identify organisms that produce the enzyme, catalase.
This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen
gas.
❖ Amylase test:
1. Principle:
Amylase testing is based on the principle of detecting the presence and activity of the
amylase enzyme in the sample. Amylase is responsible for breaking down starch into
simpler sugars such as maltose and glucose. In the test, 3 microbial samples were
cultured into an LB-starch plate medium. After incubation, add iodine to the culture
portions. Starch will react with iodine to create a blue-black color. However, if
amylase is present and active, it will break down starch, resulting in a clear zone
around microbial growth due to the absence of starch in that area. The appearance of a
clear zone indicates a positive result for amylase activity, indicating that the
microorganism is capable of producing the enzyme.
2. Protocol:

3. Result:

The iodine reacts with the starch. Thus, hydrolysis of the starch will create a clear
zone around the bacterial growth. A positive result is indicated by the formation of a
clear halo around the colonies and not develop of a dark blue to purple-blue color in
the surrounding medium after the addition of the Lugol reagent. A negative result is
indicated by no clear halo around the colonies and the development of dark blue to
purple-blue color in the surrounding medium after the addition of the Lugol reagent.
 Sample L: The color changes from dark blue to brown either no amylase is
present, or the amylase has not had sufficient time or conditions to break down the
starch.
Sample E: The slight darkening to brown suggests that there may be a small
amount of amylase present. The remaining starch could be reacting with Lugol's
solution to produce the brown color.
Sample B: The lack of color change suggests that amylase is present and has
broken down the starch, preventing the formation of the blue color with the Lugol
reagent.
4. Conclusion
Amylase test to determine whether the organism is capable of breaking down
starch into maltose through the activity of the extra-cellular α-amylase enzyme.
V. Microbial growth controlling
❖ Osmotic stress:
1. Principle:
The principle of osmotic stress involves the response of a cell or organism to
changes in the osmotic pressure of the environment. High osmotic stress, such as in
hypertonic environments, causes water to move out of the cell, leading to cell
shrinkage. Low osmotic stress, such as in a hypotonic environment, causes water to
move into the cell, potentially leading to swelling or cell rupture. In this experiment,
E. coli and Saccharomyces bacteria were used as samples to culture in 3 mediums.
The purpose is to observe and evaluate the influence of osmotic stress on the growth
and development of microorganisms.
2. Protocol:
 3. Result

LB plate: both samples E.Coli and S.cerevisiae are growing

LB + 10% NaCl plate: both samples E.coli and S. cerevisiae are not growing.
LB + 25% Sucrose plate: both samples E.Coli and S.Cerevisiae are growing,
S.cerevisiae grows better than E.coli
4. Conclusion:
In the LB medium, both microorganisms exhibit normal growth. However, in LB
medium supplemented with 10% NaCl, growth is inhibited for both due to its
hypertonic nature. In contrast, in LB medium supplemented with 25% Sucrose, S.
cerevisiae microorganisms demonstrate enhanced growth compared to E. Coli. This
preference can be attributed to yeast nature, which utilizes glucose, sucrose, and other
saccharides as nutrients, favoring its growth in this environment over E. coli.
❖ Antibiotic:
1. Principle:
The principle of antibiotics involves the use of specific substances to inhibit the
growth or kill microorganisms. Antibiotics target essential cellular processes or
structures in bacteria, fungi, or other microorganisms, disrupting their normal
functioning. This may include inhibition of cell wall synthesis, protein synthesis,
nucleic acid synthesis, or disruption of cell membrane function. It can be seen that in
this experiment, our team used E. coli bacteria as a test sample, by dividing the plate
into 3 parts and using a cotton swab containing the sample to cover the culture surface
using a multidimensional method, then placing the sterile filter disk in the middle of
each part, finally add 3 types of antibiotics (Tetracycline, Ampicillin, Kanamycin) into
the sterile filter disk to evaluate and observe whether the impact of antibiotics on
bacterial growth is strong or weak.
2. Protocol:

3. Result
 On the plate containing our group's E. coli strain, all antibiotics have a sterile
ring, and the diameter of the inhibition zone gradually decreases from Amp > kan >
Tetra: 2 out of 3 plates have a sterile ring and diameter. The inhibition zone gradually
decreases from Amp > kana > Tetra (with diameter measurements of 2.6 > 2.1 > 2.0,
respectively). The remaining 1 disc has a sterile ring with diameter measurements of
2.0 > 1.2 > 1.0, respectively.
 Based on the results above, it is possible that our team did some technical
wrong.
● Ampicillin is a beta-lactam antibiotic, belonging to the penicillin group of
antibiotics. It works by inhibiting the synthesis of bacterial cell walls. E. coli a
Gram-negative bacterium produces an enzyme called beta-lactamase. Beta-
lactamase breaks down the beta-lactam ring present in antibiotics like penicillin
and its derivatives, rendering them ineffective
● Kanamycin is indeed an aminoglycoside antibiotic that works by inhibiting
bacterial protein synthesis. It binds to the bacterial ribosome, specifically the
30S subunit, and interferes with the translocation step during protein synthesis.
This disruption ultimately leads to the inhibition of peptide bond formation and
thus prevents the bacterium from producing essential proteins for survival.
● Tetracycline is effective against Escherichia coli (E. coli) due to its mechanism
of action, which interferes with bacterial protein synthesis. Tetracycline binds
to the bacterial ribosome, specifically to the 30S ribosomal subunit, and
disrupts the process of protein synthesis. The binding of tetracycline to the
ribosome interferes with the attachment of aminoacyl-tRNA molecules to the
mRNA-ribosome complex. This interference prevents the elongation of the
polypeptide chain during protein synthesis. As a result, E. coli bacteria are
unable to produce vital proteins necessary for their cellular functions, leading
to inhibition of growth and ultimately bacterial death.
4. Conclusion:
In conclusion, antibiotics such as ampicillin, kanamycin, and tetracycline play
crucial roles in combating bacterial infections, including those caused by Escherichia
coli. Each antibiotic targets bacterial cells through distinct mechanisms, making them
effective treatments against susceptible strains of bacteria.
NOTE: The above operations must be performed in a sterile environment, next to an
alcohol lamp, disinfecting laboratory equipment, and making notes on each sample.