Group #4

Group Members: Date Performed: June 24, 2019

Rejuso, Alyssa Rae Date Submitted: June 28, 2019
Romero, Timothy
Ruiz, Kim
Sacro, Rico Rafael
Sanchez, Kristel
Santiago, Carl Vincent

Exercise No. 4
Cultivation and Isolation of Bacteria

Abstract

Handling microorganisms face exhaustive efforts to maintain an isolated and contaminant-free

cultures to simulate a closed controlled environment. Cultures are also purposive of function,
presenting specific properties microorganisms like motility and morphology. To provide insight, the
study performed streaking and plating procedures using Escherichia coli bacteria and sterilized
nutrient agar (NA) plates and tubes to understand the importance of media types, while applying
proper aseptic and inoculation practices to prevent contamination. Aseptic techniques are important
to maintain pure cultures, and flaming the brim of source cultures eliminates airborne microorganisms
from the broth, slant, stab tube media, and NA plates. Multiple ways of isolating and culturing bacteria
were done, which includes making bacterial broth to observe the oxygen requirement of bacteria,
stabs to observe motility, slants, and spread, pour, and streak plates, specifically simple streak plates
and multiple uninterrupted streak plates, to observe colony formation. After incubation, the nutrient
broth culture suggested that E. coli is an obligate anaerobe though it is said to be a facultative
anaerobe, the NA slant culture shows an echinulate structure growth, and the NA stab culture
exhibited a motility structure of filiform to papilate. The simple streak, multiple uninterrupted streak,
pour plate, and spread plate, cultures showed similar morphology of colonies of E. coli, and the
bacterial growth were deemed to be abundant that some colonies failed to grow individually from
each other. The researchers recommend that the procedures of serial dilution and inoculation be
revisited so that they would be executed more properly.

Keywords: cultivation, inoculation, streaking

I. Introduction
In cultivating microorganisms in a laboratory, a culture media must be prepared
beforehand. By definition, culture media is a substance that contains the necessary nutrients
(may be organic or inorganic) that aid in the growth of certain microscopic organisms. In the
field of microbiology, there are two broad classes of culture media that is designated,
generally, for either fastidious or non-fastidious microorganisms--defined culture media for
the former, and complex culture media for the latter.
Defined culture media is prepared through adding exact amounts of the components
needed, hence, the precise composition of the media is known. On the other hand, complex
media does not essentially require the addition of exact measurements of the constituents,
thus commonly used in cultivating various microorganisms. Although definite composition of
the media is unknown, complex culture media still poses various advantages (e.g. supporting
growth of a wide range of bacteria, digests added in the media for nutrition are commercially
available in dehydrated form, etc.) (Madigan et al. 2019).
In addition, complex culture media can be further subdivided into different types.
First, the general purpose medium. This predominantly allows the microorganisms to
multiply; noting that it contains all the essential nutrients required for growth. Subsequently,
 culture media can also be either selective or differential (or both). Selective culture media
supports the growth of a certain microorganism, while purposely inhibiting the growth of
others. Differential, on the other hand, differentiates and distinguishes different types of
bacterial organisms. This culture media mainly utilizes biochemical indicators that results into
color change (of either the media or the organism) when metabolic reactions occur during
growth. For this study, general purpose media is utilized (Paul 2013).
Given these kinds of media, they may be prepared via broths, plates , slants, or stabs;
which primarily differs in purpose. However, it is important to note that sterilization
beforehand is significant. Sterilization is a process that helps in avoiding contamination from
the environment, thus maintaining the purity of the culture. There are various ways in order
to sterilize materials needed for cultivation in the laboratory: a.) wet heat via autoclaving, b.)
dry heat via baking or flaming, c.) filtration, d.) solvents, and e.) radiation. Each method
undoubtedly displays various advantages over the other, however, in this case, autoclaving
was the primary method of sterilization.
Autoclaving is a method that uses pressure, temperature, and steam in order to kill
bacteria, spores, and germs that may cause contamination to the bacterial culture. In addition,
this moist heat procedure uses pressurized steam that has high latent heat, which as a result,
holds seven (7) times more heat than water at the same given temperature. Noting this, it
allows rapid transfer of heat and better penetration to thick materials, thus displays a great
advantage compared to other sterilization techniques (Madigan et al. 2019).
It is established that sterilization is an important process for cultivation of bacteria.
Therefore, it is necessary to understand that sterilization while isolating bacteria is a
significant process to be done. With this, aseptic technique has been introduced in the field
of microbiology.
Aseptic technique is a multi-step procedure of flame sterilizing and ethanol-
immersing in order to maintain the purity of bacterial cultures. This is often done when
transferring axenic bacterial cultures to different culture media. With this, it also ensures that
contamination from the environment and cross-contamination from other samples are
avoided
Keeping in mind the aseptic technique, there are various means into isolating bacterial
samples. For instance, the isolation in tubed media (e.g. slant tubes, stab tubes, and broth
tubes) and in plated media (e.g. simple streak plating, multiple uninterrupted streak plating,
spread plating, and pour plating). These isolation techniques aid in determining bacteria’s, in
this case Escherichia coli, colony morphology and motility. In addition, for most of these
methods, pure cultures are used. However, for spread plating and pour plating, diluted (10−5 )
bacterial culture, through serial dilution, is utilized. Serial dilution is a stepwise process that is
done in order to avoid excessive bacterial growth on an agar plate (Society for General
Microbiology 2006).
In order to successfully observe, cultivate, and isolate a bacterial culture inside the
laboratory, there are fundamental steps to be followed, especially in terms of sterilization.
Hence, the study has the following objectives:
● To perform aseptic technique in inoculating bacteria in various forms of culture
media; and
● To perform different isolation methods for bacteria.

II. Materials and Methodology

A. Aseptic Technique
Aseptic Technique. The work area was disinfected with 70% alcohol. Hands were
washed thoroughly and dried with clean paper towels. The alcohol lamp was
positioned accordingly to prevent hampering of movement and lighted using matches
 or a lighter. A beaker filled one-third with ethanol was provided for sterilization of
laboratory equipment (ADMU n.d.).

B. Aseptic Transfer of Bacterial Culture

Aseptic Transfer of Bacteria. The inoculating loop was dipped in ethanol solution then
flame-sterilized. The loop was cooled by the flame of the alcohol lamp. The source E.
coli broth culture was shaken gently and opened near the flame. The opening of the
broth culture was sterilized via passing through the flame. Before extracting E. coli
growth, the loop was slightly dipped into the broth to check whether the loop is still
too hot. Once appropriately checked if cooled, E. coli was extracted from the broth
culture using the inoculating loop. The opening of the source broth culture was flamed
prior closing. The bacterial culture was inoculated to a suitable medium, with
appropriate inoculation techniques described in the following parts of the paper
(ADMU n.d.).

C. Inoculation of Test Tubes

Inoculation of Nutrient Broth Tube. Three sterilized nutrient broth tube media were
procured and labeled accordingly. An inoculating loop was flame-sterilized and
cooled, and then dipped into a gently shaken tube containing bacterial culture. The
nutrient broth tube was held with a free hand. Following the aseptic technique, the
cotton plug was removed by the pinky finger and placed properly while passing the
opening of the nutrient broth tube through flame. The inoculum was then transferred
by dipping and mixing the inserted inoculating loop. The inoculating loop was then
flame-sterilized again and cooled. The nutrient broth tube was then sealed with the
cotton plug after passing its opening through flame. They were then incubated at 37ºC
for 24 hours (ADMU n.d.).

Inoculation of Nutrient Agar Slant Tube. Three sterilized NA slant tube media were
procured and labeled accordingly. An inoculating loop was flame-sterilized and
cooled, and then dipped into a gently shaken tube containing bacterial culture. The
NA slant tube was held with a free hand. Following the aseptic technique, the cotton
plug was removed by the pinky finger and placed properly while passing the opening
of the slant tube through flame. The inoculating loop was then inserted into the tube
and the inoculum was gently streaked along the surface of the NA with a zigzag
motion. The inoculating loop was then flame-sterilized again and cooled. The NA slant
tube was then sealed with the cotton plug after passing its opening through flame.
They were then incubated at 37ºC for 24 hours (ADMU n.d.).

Inoculation of Nutrient Agar Stab Tube. Three sterilized stab tube media were
procured and labeled accordingly. An inoculating needle was flame-sterilized and
cooled, and then dipped into a gently shaken tube containing bacterial culture. The
NA stab tube was held with a free hand. Following the aseptic technique, the cotton
plug was removed by the pinky finger and placed properly while passing the opening
of the NA stab tube through flame. The inoculating needle was inserted and stabbed
into the center of the agar. The inoculating needle was then flame-sterilized again and
cooled. The NA stab tube was then sealed with the cotton plug after passing its
opening through flame. They were then incubated at 37ºC for 24 hours (ADMU n.d.).

D. Streak Plating Technique

Simple Streak Plating. NA plates were prepared. An inoculating loop was flame-
sterilized and cooled, and then dipped into a gently shaken tube containing bacterial
 culture. The NA plate was held with a free hand, with the bottom of the plate was
supported with the last three fingers and the cover plate with the thumb and index
finger. Following the aseptic technique, the plate was briefly passed through the
flame, and using the thumb and index finger, the cover was lifted, keeping the opening
of the lid as small as possible. The inoculum was then streaked on the plate in a zigzag
motion. The inoculating loop was then flame-sterilized again and cooled. The
inoculated plates were then sealed with parafilm, and wrapped upside down with
paper and then labeled. They were then incubated at 37ºC for 24 hours (ADMU n.d.).

Multiple Interrupted Streak Plating. NA plates were prepared. The NA plate was held
with a free hand, with the bottom of the plate was supported with the last three
fingers and the cover plate with the thumb and index finger. An inoculating loop was
flame-sterilized and cooled, and then dipped into a gently shaken tube containing
bacterial culture. Following the aseptic technique, the plate was briefly passed
through the flame, and using the thumb and index finger, the cover was lifted, keeping
the opening of the lid as small as possible. With the plate mentally divided into three
parts, the inoculum was then streaked on one side of the plate in a zigzag motion and
the lid was closed. The inoculating loop was then flame-sterilized again and cooled.
Turning the plate 120°, the cover was slowly lifted again, and inoculating loop was
streaked in a zigzag motion starting from the last streak of the first section. New
inoculum was not obtained from the tube of bacterial culture. The plate was then
covered, and the inoculating loop was flame-sterilized again and cooled. This process
was repeated one last time for the third sector of the NA plate. The inoculated plates
were then sealed with parafilm, and wrapped upside down with paper and then
labeled. They were then incubated at 37ºC for 24 hours (ADMU n.d.).

E. Spread Plating Technique

Serial Dilution. Three screw cap tubes were prepared. The first screw cap tube was
filled with 9mL of 0.85% saline (NaCl) solution, and the second and third screw cap
tube with 9.9mL of the same solution. Using a micropipette, the first dilution factor
(10-1) was made by adding 1mL of the culture into the first screw cap tube which was
then mixed using a vortex mixer. To note, new micropipette tips were used after every
transfer. The second dilution factor (10-3) was made from the first dilution by
transferring 0.1mL into the second screw cap tube and mixed using the vortex mixer.
The third and final dilution factor (10-5) was made by transferring 0.1mL of the second
dilution factor to the third screw cap tube and again mixed using the vortex mixer
(ADMU n.d.).

Spread-plating technique. A sterilized dry NA plate was procured. The mouth or the
opening of the NA plate was flamed before micropipetting 0.1 mL of the 10 -5 dilution
into the middle of the NA plate. The spreader/L-shaped rod was sterilized by briefly
submerging it in 70% EtOH. The spreader was briefly heated and then cooled beside
the flame. Using the newly sterilized spreader, the bacteria was evenly spread all over
the center of the NA, while avoiding the edge of the NA plate. To do this evenly, the
plate was rotated manually. Then, the NA plate was closed, flamed by the mouth, and
was left to dry completely before sealed with parafilm and wrapped invertedly. The
wrapped petri dish was labelled accordingly. The spreader was flamed and dipped
 again in EtOH for sterilization purposes. As for the NA plate, it was inverted while
incubated at a temperature of 37°C for at least 24 hours (ADMU n.d.).

F. Pour Plating Technique

Pour Plating Technique. Properly label a sterile, dry, and empty petri plate. Before
opening the petri plate, the mouth of the opening was heated. Using a micropipette,
1mL of the 10-5 dilution from the serial dilution was placed aseptically into the middle
of the petri dish. 15-20mL of molten NA, with a temperature of around 45-50C, was
then poured aseptically into the area where the dilution was placed. Immediately,
the petri dish was covered and flamed on the mouth. To mix the inoculum with the
agar, the plate was moved on the table in figures of 8 repeatedly, making sure there
are no spillage. Once the agar has cooled, parafilm was used to seal the NA plate, and
wrapped it invertedly. Lastly, the NA plate was Incubated for around 24 hours at a
temperature of 37°C. Once incubation is done, it was refrigerated invertedly (ADMU
n.d.).

III. Results and Discussion

Importance of Aseptic Technique. In cultivating bacterial samples, it is important to grow

cultures that does not contain any growth from other organisms; hence, aseptic technique
was introduced. As discussed above, aseptic technique is a series of steps that primarily
involves flame sterilizing. In manipulating bacterial cultures, it is important to execute this
process in order to maintain the purity of the culture when transferring it to a new culture
media. In addition, this also aids in avoiding contamination from the environment, and cross-
contamination from other samples (Madigan et al. 2019).

Heating the Lip of a Test Tube. Before opening the test tubes containing either agar of broth,
the mouth of the test tube must be flamed. This goes the same for NA plates and petri dishes.
Convection currents are created when the lip of the test tube is being heated, forcing the air
inside the tube to escape. Air borne contaminants are then blocked by these convection
currents (Vlab.marita, 2011). Due to the heat coming from the bunsen burner, the air around
the working area also rises, lowering the risk of airborne bacteria from contaminating the
culture.

NUTRIENT BROTH
 Figure 1. NA Broth Exhibiting E. coli Bacterial Growth
After 18-24 Hours of Incubation at 37 Degrees Celsius
Description:
Figure 1 shows the result of the nutrient broth 24 hours after incubating at a temperature
of 37C. Solvents are found at the bottom of the broth, which may suggest that the
microorganism used, E. coli, is an obligate anaerobe (See Appendix A). However, the test
tube also appeared quite cloudy. The cloudiness may signify that there was an increased
number of microbes found in the nutrient broth. However, E. coli is said to be a facultative
anaerobe, meaning they can grow in both oxygen rich and oxygen poor environments. Since
the ability to develop in changing environments is a characteristic that facultative anaerobic
bacteria has, it can also be said that the organism Escherichia coli is one (von Wulffen et al.
2016).

NUTRIENT AGAR SLANT

 Figure 2. NA Slant Exhibiting E. coli Bacterial Growth
After 18-24 Hours of Incubation at 37 Degrees Celsius

Description:
The NA exhibited above is slanted at a 30 degree angle and the cultured E. coli inoculum
demonstrates an echinulate structure on the medium after 18-24 hours of incubation at 37
degrees Celsius (Appendix B).

NUTRIENT AGAR STAB

 Figure 3. NA Stab Exhibiting E. coli Bacterial Growth and Motility
After 18-24 Hours of Incubation at 37 Degrees Celsius

Description:
The NA stab shown above demonstrates motility of the cultured E. coli inoculum through
the stab zone located at the center of the media and ¾ below the surface of the media.
Qualitatively, the structural mode of motility demonstrated is filiform to papilate (Appendix
C).

POUR PLATE

Pour Plating. A pour plate is a technique in which a small amount of inoculum from broth
culture (i.e. 1 mL of 10-5 serial dilution) is added by micropipette to the center of a Petri dish.
Molten and cooled agar medium is then poured into the Petri dish containing the inoculum.
The dish is gently rotated or mobilized by dragging it in an 8-motion to ensure that the culture
and medium are thoroughly incorporated and the medium covers the plate evenly.
Afterwards, the pour plate is incubated for 18-24 hours at 37 degrees Celsius to promote
bacterial growth. Pour plates tolerate bacteria to grow both on the surface and within the
medium. Majority of the colonies grow within the medium and are small in size and may be
confluent. The minority that grow on the surface are of the same size and appearance as those
on a streak plate. If the dilution and volume of the inoculum, usually 1 , are determined, the
viable count of the sample, i.e. the number of bacteria or clumps of bacteria, per cm3 can be
determined. The dilutions selected must be appropriate to produce between 30 and 100
separate countable colonies (Society for General Microbiology 2006).
 Figure 4. Pour Plate Exhibiting E. coli Bacterial Growth
After 18-24 Hours of Incubation at 37 Degrees Celsius

Description: E. coli inoculated through pour plating was shown to grow scattered
throughout the medium. The culture was observed to be dull, dry, greyish-white color,
translucent, and has clumped colonies in various sizes. The colonies were noticed to grow
in a circular or irregular form, with raised elevation, and having an entire margin (Appendix
D).

SIMPLE STREAK PLATE

Figure 5. Simple Streaking Plate Exhibiting E. coli Bacterial Growth

After 18-24 Hours of Incubation at 37 Degrees Celsius

Description: E. coli bacteria inoculated through simple streaking was observed to grow into
 colonies on the center and towards one side. The simple streaking was unsuccessful in
distributing the cells throughout the agar evenly. Additionally, though streaked only on the
center and not the edges, E. coli still proliferated on the edge of the NA. The culture was
observed to be dull, dry, greyish-white color, translucent, and has clumped colonies in
various sizes. The colonies were noticed to grow in an irregular form, with raised elevation,
and having an entire margin (Appendix D).

Streak Plating. Streak plating is a technique utilized to isolate a pure strain from a single
species of bacteria. Samples can be obtained from the resulting colonies and a pure
microbiological culture can be grown on a new plate so that the organism can be empirically
observed, identified, or examined. This demonstrates the progressive dilution of a pure
culture of bacteria by systematically streaking them over the surface of the agar in a Petri dish
to obtain and grow isolated colonies or quantity of bacteria; these enable colonies to grow
well separated from each other. If the agar surface grows microorganisms which are all
genetically similar, the culture is then considered as a pure microbiological culture. Streaking
is performed using a sterile inoculation loop and aseptic techniques are integrated to maintain
pure cultures and to avoid contamination of the growth medium. Simple streaking is done by
spreading the inoculum in a zigzag manner on the non-divided and non-sectored agar plate
surface. In terms of multiple interrupted streaking protocol, the inoculation loop is first
sterilized by passing it through a flame. When the loop has cooled down, it is dipped into an
inoculum or a broth containing the pure culture of bacteria. The inoculation loop is then
dragged across the surface of the agar plate back and forth in a zigzag motion until 30% of the
plate has been covered. The loop then is re-sterilized through the flame and the plate is turned
120 degrees. Starting from the previously streaked section, the loop is dragged through it
twice or thrice continuing the zigzag pattern. The procedure is reiterated once more being
careful to not touch the previously streaked sectors. The loop gathers fewer and fewer
bacteria until it gathers just single bacterial cells that can grow into a colony. The plate should
exhibit the most dense growth in the first section. The second section will have less growth
density and a few isolated colonies, while the final section will have the least amount of
growth density and many isolated colonies. (Black and Black 2014).
Similarly, while streaking in successive sectors of the agar plate, the inoculum is
diluted to the point where there is only a single bacterial cell deposited every few millimeters
on the surface of the agar plate. When these lone bacterial cells divide and emerge to several
of new bacterial cells, an isolated colony is established. Pure cultures can be produced by
selecting well isolated colonies and re-streaking these on fresh agar plates (Medical
Microbiology Guide 2016).
Depending on the type of bacterial strain, the plate may then be incubated, usually
for 18-24 hours at 37 degrees Celsius, to allow the bacteria to reproduce. At the end of
incubation, there should be sufficient bacteria to form visible colonies in the areas touched by
the inoculation loop. From these colonies, single bacterial species can be identified based on
their morphological differences, and then subcultured to a new media plate to yield a pure
culture for further examination (Black and Black 2014).

MULTIPLE INTERRUPTED STREAK PLATE

 Figure 6. Multiple Interrupted Streaking Plate Exhibiting E. coli Bacterial Growth
After 18-24 Hours of Incubation at 37 Degrees Celsius
Description: E. coli bacteria inoculated through multiple interrupted streaking was
observed to unsuccessfully grow individual colonies, especially in the third round of
streaking. The culture was observed to be dull, dry, greyish-white color, translucent, and
has clumped colonies in various sizes. The colonies were noticed to grow in an irregular
form, with raised elevation, and having an entire margin (Appendix D).

SPREAD PLATE

Spread Plating. Spread plate technique is a method practiced to plate a liquid sample for the
purpose of isolating, observing, or quantifying the bacteria present in that sample. A perfect
spread plate technique will manifest visible and isolated colonies of bacteria that are evenly
distributed in the plate and are quantifiable. The sample is then diluted in 10 fold serial
dilutions and plated in an appropriate medium. Correspondingly, 0.1 mL of the 10-5 serial
dilution of the sample is obtained via micropipette and spread on top of the surface using an
L-shaped glass spreader. The spread plate will then be incubated for 18-24 hours at 37 degrees
Celsius for bacterial growth. Following incubation, the number of colonies present in the plate
are quantified. Assuming that each organism provides a single colony, the number of total
bacteria present in a sample are calculated (Rijal 2017). These plates should instigate a dense,
often confluent growth of culture spread evenly over the surface of the growth medium. This
implies that these plates can be utilized to examine the sensitivity of bacteria to many
antimicrobial substances. Additionally, the spread plate can be utilized for quantitative work
or colony counts. If the dilution and inoculum volume are known (i.e. 0.1 cm3), the viable count
of the sample (i.e. number of bacteria per cm3) can be determined. The dilutions established
must be appropriate to produce between 30 and 100 separate countable colonies (Society for
General Microbiology 2006).
 Figure 7. Spread Plate Exhibiting E. coli Bacterial Growth
After 18-24 Hours of Incubation at 37 Degrees Celsius

Description: E. coli bacteria inoculated through spread technique was observed to

unsuccessfully grow individual colonies, as the grown bacteria is shown to be heavily
clumped together with no individual colonies present. Additionally, though only poured on
the center, E. coli proliferated at the edge of the NA plate. The culture was observed to be
dull, dry, a greyish-white color, translucent, and has clumped colonies in various sizes. The
colonies were noticed to grow in an irregular form, with raised elevation, and having an
entire margin (Appendix D).

Inverting Agar Plates. Before incubation, all NA plates should be wrapped and incubated
invertedly for at least 24 hours at a temperature of 37°C. During incubation, NA plates are
invertedly placed because of the risk of contamination present due to many reasons. The
samples may be contaminated as warm incubation leads to condensation which then allows
the microbes in an uninverted agar plate to spread and combine with other colonies. This
occurs as water which drips on the organisms allows them to move across the plate (Malesky,
2017). Another risk is that these NA plates or the lids of the dishes may not have been properly
cleaned which can also lead to the contamination of microbes to the samples. Lastly, the
evaporation rate of the water used to grow bacterial samples increases if an agar plate is
placed uninverted, and this evaporation may again lead to condensation (Choudhary, 2015).

Consequences of a Wet Surface in Isolation. In some instances, water vapor are produced by
microbes as by-products. As the bacterial cells multiply and bacterial colonies grow, water
vapor will be produced. If the plates are in a normal upright position, then the vapor will
accumulate on the top lid. Hence, once enough water vapor is accumulated and condensed
at the top lid, drops of water will drop down onto the bacterial colonies, resulting to washing
them away into one big mixed colony, leaving the plate with very minimal well isolated
colonies. Nonetheless, plates must be inverted so that water droplets can’t ruin the isolated
single colonies of bacteria on the plates (Ranjan 2013).

IV. Conclusions and Recommendations

 The researchers used different methods and culture media in isolating and cultivating
E. coli. These methods and culture media include nutrient broth, NA slant, and NA stab in test
tubes and simple streak, multiple uninterrupted streak, spread, and pour plating. In every
method and techniques, proper aseptic practices were followed such as flame-sterilizing the
inoculating tool and passing the opening of the glasswares through flame. All of the cultures
were then incubated at 37ºC for 18-24 hours.
Upon inspection after incubation, the nutrient broth culture suggests that E. coli is an
obligate anaerobe though it is said to be a facultative anaerobe (von Wulffen et al. 2016). The
NA slant culture shows an echinulate structure growth of E. coli. The NA stab culture exhibited
the motility of E. coli with a motility structure of filiform to papilate.
The simple streak, multiple uninterrupted streak, and pour plate culture shows
colonies of E. coli with a circular or irregular form, with raised elevation, and having an entire
margin. However, the inoculum was not evenly inoculated in simple streak technique thus not
allowing the even growth of E. coli. Additionally, the bacterial growth in simple streak, multiple
uninterrupted streak, and spread plate were deemed to be abundant that some colonies
failed to grow individually from each other. In spread plate, the grown bacteria is heavily
clumped thus failed to show individual colonies. These errors might be caused by improper
inoculation and serial dilution. It is recommended that the procedures of serial dilution and
inoculation be revisited so that they would be executed more properly.
In this experiment, the researchers failed to properly grow and culture bacteria in the
NA plates. With this, the researchers understood the importance of strictly following protocols
and procedure as slight errors might cause problems to the results of the experiment.

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Appendix A

Analysis of Results: Nutrient Broth

 Appendix B

Analysis of Results: Nutrient Agar Slant

 Appendix C

Analysis of Results: Nutrient Agar Stab

 Appendix D

Analysis of Results: Agar Plates