Group 2 Laboratory Activity 1 & 2

Date performed: September 19 & 21, 2023

Group Members:

N. Bagsic, J. Bantayan, G. Bermas, B. Borja, N. Calubad

Microbial Growth Media and Gram-Staining as Introduction to Laboratory

Activities

Abstract

Microbial growth media and gram-staining have been introduced to students for
better understanding of the topic through learning through experience. This laboratory
activity became an eye-opener for the students to relate the knowledge from the books
to the practical way of doing it. In the first activity, the growing of microbes, the students
prepared 42g of nutrient agar and distilled water using a pan and stove for 15 minutes
and put it in an pressure cooker to sanitize it. After letting it cool down, 1/3 of the
nutrient agar is poured into the petri dish. After a few minutes, a cotton tip applicator
with the sample of growing microbes from the urinal bidet is used to streak plate it to the
surface of the agar in the petri dish. Finally, after 48 hours, the samples are viewed. In
the activity conducted, based on the chemical and physical characteristics of their cell
walls, the gram stain is a differential staining technique used to classify bacteria as
gram-positive or gram-negative. The steps in gram-staining are crystal violet as the
primary stain, iodine as the mordant, ethanol as the acetone or decolorizer, and safranin
as the counterstain. A variety of staining and decolorization procedures are used to
separate the microorganisms. In contrast to gram-negative cells, which stain red to pink,
gram-positive cells have a purple stain. Unfortunately, the results show no microbial
growth after 2 days because the nutrient agar is expired even though the quantity is
doubled. There are no bacteria viewed under the microscope after gram-staining the
fermented milk drink Yakult as a sample granted that the students followed the
procedures in gram-staining respectively. However, as an alternative, the students
researched on the internet the microbes that can be found in restrooms, which are
Escherichia coli (E. coli), and this was chosen by the group as a sample. In gram-
staining, Lacticaseibacillus paracasei Shirota (L. paracasei Shirota) is the bacteria that
Yakult contains. The group found out by browsing that this bacterium is gram-positive
and facultative anaerobic.

I. Introduction

The development and differentiation of microorganisms are encouraged and

supported by a mixture of substances known as a microbial culture medium. The
ingredients in culture media include nutrients, energy sources, growth-promoting agents,
minerals, metals, buffer salts, and gelling agents (for solid media). Gram-staining is a
method frequently used to distinguish between two sizable groups of bacteria based on
the distinct components of their cell walls. By staining these cells red or violet, the Gram
stain method distinguishes between Gram positive and Gram negative groupings. The
crystal violet used to stain the cells of gram-positive bacteria is retained by the presence
of a thick layer of peptidoglycan in their cell walls. In contrast, Gram negative bacteria
stain red because their peptidoglycan walls are thinner and don't hold on to the crystal
violet during the decolorizing process, causing them to stain red.

Aseptic technique is the method that uses target-specific practices and

processes under adequately controlled conditions to decrease contamination from
germs. Conducting research in the area of microbiology requires a certain level of
laboratory proficiency. The cultures have not been contaminated by innate or acquired
bacteria from the environment as a result to a proper aseptic procedure. It is possible to
drastically lower/minimize the risk of infection by using the right aseptic approach. Pure
stock cultures and single spore cultures are frequently kept while being transferred onto
fresh media using aseptic procedure. Effective aseptic practices guard against
microorganisms being mistakenly released into the environment or contaminating lab
users. The goals of aseptic techniques are to learn aseptic technique in the field of
microbes, prevent the contamination of lab cultures by undesirable microbes, subculture
(transfer cultures from one media by inoculating into another media), isolate pure
cultures from mixed cultures, and prevent lab microbes from spreading in the
environment.

This activity shows the process of growing microbes on a medium and gram-
staining. Also, aseptic technique goes along with every exercise during this laboratory
activity. This will also help the students practice and experience how microbes grow and
develop colonies. The student will also determine the microbes based on their
morphology and whether they are gram-positive or gram-negative. This activity helped
the students better understand the subject matter, especially in the future.

II. Materials and Methods

In the first activity, which is the microbial growth medium, the students prepared
42 grams of nutrient agar and 850 mL of distilled water and put it in the beaker while
wearing gloves, a mask, a lab gown, and other proper gear while doing laboratory
activities. However, something unexpected happened that the making failed. As an
alternative, a pan and stove are used to prepare the agar. Gerald used a cotton tip
applicator and the chronicle tube to get the sample, specifically the urinal or bidet in the
restroom of CBSUA-Admin. After letting it cool down, Joyce volunteered to pour the
agar into the Erlenmeyer flask, covered with foil, and put in the pressure cooker for 10–
15 minutes. While letting it cool down, Bianca sanitize the table using disinfectant to
avoid cross-contamination. The nutrient agar was then poured 1/3 into the petri plates,
which were already labelled with their sample, group number, and the date and time it
was performed. After leaving it for a while, it is time to use the streak plate technique on
the surface of the nutrient agar and use their sample. After that, the samples are put in
the incubator at 37°C. After 48 hours, finally the viewing of microbial growth in the
student’s sample.
 The cells can be stained once a loop full of an overnight culture has been heat
fixed. The first stain employed is a primary stain called crystal violet. All organisms will
be stained by this cationic substance since cells have a negative charge. Flood with
crystal violet stain, let it sit for a minute, and then rinse with water. Following rinsing,
gram’s iodine is used as mordant to help retain the primary stain. Flood the stain with
iodine solution, apply it for a minute, and then rinse it with water. Ethanol alcohol (EtOH)
is used during the second rinse. One of the roles of EtOH is to reduce the size of the
pores in the peptidoglycan layer.

The gram-positive cell is then sealed off with the crystal violet-iodine
combination. In comparison to gram-positive species, the larger pores and thinner the
peptidoglycan in gram-negative organisms are not altered to the same an extent.
However, the gram-negative organisms’ outer membrane is similarly eliminated by the
EtOH. In essence, the gram-negative cells are successfully rendered colourless by the
EtOH rinse. Counterstain the slide with safranin for 30 seconds until the gram-positive
are stained purple, then rinse with water. In order to colour the colourless gram-
negatives, a counterstain is used. These will have a purple colour. Now that the slide is
air-dried, make sure it will not be destroyed or contaminated by placing it between book
pages. Remove slide and view organisms using the oil immersion objective of the
microscope.

III. Results and Discussion

Microbial Growth Media

A liquid or gel that is intended to facilitate the growth of microorganisms is known

as a culture medium or growth medium. There are several media types that are suited
for growing various cell types. Culture medium refers to the substances or materials
necessary for microorganism growth. Figure 1 shows a petri dish with a 1/3 of nutrient
agar that consist of labelled sample, including date and time, group name, and the place
where the sample is from.
 Figure 1. Shows labelled bottom of agar plate. Includes group name, lab section,
date, media type (NA for nutrient agar) and what was sampled. Note: Make sure
labeling of your plates is clear and complete.

When agar is mixed with water it solidifies and forms a gel, it liquefies at boiling
100°C and does not resolidify until it cools to 42°C, which is called the gel point.
Although different agars vary considerably in their physical properties, the usual melting
point is 97-100°C. If the agar is too hot and it is poured into plates, the bacteria in the
sample have the possibility to die. When the bacteria died it could not be use as sample
because it could result to a negative Gram stain or no organism will be seen at the
microscope when viewed and no growth in the sample will happen because of the hot
temperature. Furthermore, the lids will develop an excessive amount of condensation if
the agar is excessively hot. The medium may become lumpy after solidifying if the agar
is put onto a plate while it is still too cold. They were useless because the medium in the
container would begin to solidify.

It is important for the culture media to be sterilized before they can be used for
establishing pure cultures of microorganisms, because through sterilization the growth
of other microorganism will be eliminated. Solid medium is usually made by adding a
solidifying agent to a broth medium. The most common solidifying agent is agar, a
substance obtained from marine algae and available in dried purified form. Due to its
melting characteristics and the lack of nutritional value for the vast majority of bacteria,
agar is an excellent solidifying agent for microbial media.

Inoculation is the deliberate introduction of microorganisms into an

uncontaminated growing environment. To stop bacteria from spreading from the
environment into a culture media, aseptic techniques are used. Care and attention are
needed when using these methods. First, to prevent contamination, always use gloves
and a mask, clean the work area before and after usage, limit the time that cultures and
growth media are exposed to the environment, and avoid touching or breathing into
sterile culture media. The inoculating loop and Petri dish are disposable, thus there is
no need to place it in an autoclave to sanitation. The tube caps should be held in the
hand while working with tubes rather than being placed on the tabletop. The tube caps
should always be held in the student's hand while performing an injection when working
with tubes. So, in streak plating, streak the surface of an agar plate (just a touch) with
the inoculating loop.

Open your petri dish and set out. Even when wearing gloves, be careful when
handling the interiors as you could pick up microorganisms that will contaminate your
sample. Place the agar bottle in the glass with the lid off and some boiling water on a
suitable container. Take care not to immerse the bottle let water get inside of it. Once
the agar has liquefied, carefully remove the bottle from the glass with the glove on it
because it will be hot. Give it some time to cool and rest so that you can securely pour it
into petri dish. You only need to use a thin layer to cover the entire bottom of the dish.
The next step is to take your sterile swab, open it up, and rub it on the area you wish to
test. You should only test one surface at a time since you don’t want to cross-
contaminate. Next, take your test swab and gently do the streak plate technique. You
don’t need to press firmly; as long as they make contact. Now cover your petri dish with
its lid and tape it securely; do not open it once more.
 Figure 2. Shows the Streak Plate Technique.

Unfortunately, there has been no microbial growth in the medium, which implies
no colonies have developed. The group nevertheless managed to provide a sample of
bacteria from the internet and examined its habitat, growth pattern, cultural
requirements, and colony characteristics after two days. One of the microbes that can
be found in restrooms is the Escherichia coli (E. coli). Since there was no growth in the
medium, the group has decided to use E. coli bacteria as their sample. Figure 3
illustrates how E. coli on nutrient agar medium creates enormous, thick, greyish white,
wet, smooth, opaque, or translucent disc-like colonies. Colonies found in fresh isolation
have smooth forms (S) that are simple to emulsify in salt water. The rough forms (R) of
colonies found in older cultures, having dull surfaces that are frequently auto-
agglutinable in saltwater. Repeated subcultures cause S-R variation, which is linked to
the loss of virulence and typically of surface antigens.
 Figure 3. Shows the features of colonies after 2 days. (source)

Since there is no bacterial growth developed in the culture medium the group 2
then look for the same bacteria that could find at the same environment. What they
found is the E. coli which they also looked for its habitat, growth pattern, cultural
requirements, and colony characteristics. The bacillus known as E. coli as gram-
negative, straight, rod-shaped, non-sporing, non-acid, and can exist alone or in pairs.
Typically rod-shaped, cells range in size from 1-3 m 0.4-0.7 m (micrometre) and are 1 m
long 0.35 m broad, and 0.6-0.7 m in volume. Few strains are not motile, and this one is
because pf the peritrichous flagellar configuration. The best known E. coli is active at
37°C (98°F), certain laboratory strains can grow as high as 49°C (120.2°F). When the
conditions are right, reproduction can happen in a little as 20 minutes. Both motile and
non-motile fimbriae strains are known. Some E. coli cells include a polysaccharide
capsule. The E. coli using negative-staining techniques, which result in a brilliant halo
over dark background, coli capsules can be seen clearly. They only have one or two
peptidoglycan layers in their thin cell wall.

Gram-Staining

Since no bacteria had grown in the medium, group 2 provided Lacticaseibacillus

paracasei Shirota (L. paracasei Shirota) as a substitute. In order to determine whether
the cells are gram-positive or gram-negative, group 2 the looked for an interpretation.
What they discovered was that lactobacilli are non-spore-forming, gram-positive,
microaerophilic, or facultatively anaerobic rods.

When grown on solid media (and even some liquid media), bacteria can take on
distinct shapes and growth patterns. Colony morphology describes the overall
appearance of the colony and varies according to whole colony by its form: a.)
punctiform or small dots; b.) round like a circle; c.) filamentous with stringy extensions;
d.) rhizoid that appear like tree roots. Elevation varies in terms of; a.) Convex; b.) Flat; c.)
Raised; d.) Growth into medium; e.) Pulvinate. Margin or edges: a.) smooth entire; b.)
irregular; c.) rhizoid; d.) lobate; e.) filamentous; f.) curled) as shown in Figure 4. For
better understanding, Figure 5 shows the morphology of colonies.

Figure 4. A sample of colony morphologies based on margin, form, and elevation

(Modified after Willey et al., 2011).

 Figure 5. Providing a pictorial guide to better understand colony morphology.

Gram-positive bacterial species have a thick layer of peptidoglycan outside of the

plasma membrane. Figure 6 show the structure of gram-positive bacterial cell which
shows that it has a plasma membrane and a thick layer of peptidoglycan. Furthermore,
Figure 7 indicates the image of gram-positive bacteria that can be viewed under the
microscope. Gram- positive bacteria show a purple colour.

Figure 6. Shows the gram-positive Figure 7. Shows the gram-positive

bacterial cell. bacteria under the microscope

Gram-negative bacterial species have a thin layer of peptidoglycan outside of the

plasma membrane with an outer membrane outside of the peptidoglycan layer.
Embedded in the outer membrane of Gram-negative cell walls are lipopolysaccharides
(LPS). Figure 7 show the structure of gram-negative bacterial cell which shows that it
has a plasma membrane and a thin layer of peptidoglycan and also its outer membrane.
Furthermore, Figure 8 indicates the image of gram-negative bacteria that can be viewed
under the microscope. Gram-negative bacteria show a pink or red colour.

Figure 6. Shows the gram-negative Figure 7. Shows the gram-negative

bacterial cell. bacteria under the microscope

The results from the other sample, in which they utilized Yakut in gram-staining to
determine if the cell is gram-positive or gram-negative, were obtained since the
laboratory activity, titled The Bacterial Growth, was not successful. The gram positive
Lacticaseibacillus paracasei Shirota (L. paracasei Shirota) microbe, which is known as
the microbe in the sample, can be recognized by its purple or blue hue, which indicates
that it is a gram-positive bacteria are L. paracasei Shirota are typical part of the oral,
digestive, and female genital microbiota in humans. They are well-known probiotics that
help to strengthen people’s defenses.
IV. Conclusions and Recommendations

The first exercise, which is about microbial growth media, has numerous
procedures, and each of them needed extra careful handling. The group learned
different things about processing media used for microbial growth, not only the specific
steps but also the alternative ways. A large variety of non-fibrous organisms can grow in
nutrient agar, which is an all-purpose medium. The reason why nutrient agar is so
popular is that it encourages the growth of many different kinds of bacteria and fungi
and contains many of the nutrients required for bacterial growth. The alternative
microbe browsed by the students, specifically the E. coli which can be found in the
same environment where the first sample is found, is gram-negative, straight, rod-
shaped, non-sporing, non-acid, and can exist alone or in pairs. Typically rod-shaped,
cells range in size from 1-3 m 0.4-0.7 m (micrometre) and are 1 m long 0.35 m broad,
and 0.6-0.7 m in volume. Few strains are not motile, and this one is because of the
peritrichous flagella configuration. In the second activity, the gram-staining, the students
also learned each step that will help to analyze whether the sample is gram-positive, if it
is purple or gram-negative, if it is red or pink. Group 2 provided Lacticaseibacillus
paracasei Shirota (L. paracasei Shirota) as a substitute. It is gram-positive with a purple
bacterial cell. In conclusion, performing laboratory activities greatly helped the students
learn the process of growing and gram-staining the microbes through experience. The
group recommends checking the laboratory apparatus first before using it because
safety should be prioritized. Although wearing proper lab attire is required for protection,
doing laboratory activities should be done with care. Also, proper sanitation is important
to prevent contamination in both microbial growth and gram-staining.
V. References

Merck KGaA, Darmstadt, Germany and/or its affiliates. (2023). Microbial Culture Media.
[Link]
medi

Biotrend. (n.d.). Nutrient agar - Non-selective solid media for microbiology Biotrend.
[Link]
[Link]#:~:text=Nutrient%20agar%20is%20a%20general,for%20the%20growth%20o
f%20bacteria.

Gram staining. (n.d.). Microscopy.

[Link]

Siddiquee, S. (2017). The basic concept of microbiology. In Fungal biology (pp. 1–15).
[Link] Growth Media.

Basavaraju, M., & Gunashree, B. (2023). Escherichia coli: An Overview of Main

Characteristics. In IntechOpen eBooks. [Link]

Dahal, P. (2023). Streak Plate Method- Principle, Types, Methods, Uses. Microbe Notes.
[Link]
limitations/