Culturing Microorganisms From Environmental Samples

Cagayan State University - Lal-lo campus | College of Teacher Education
SCI 70 Microbiology and

Parasitism ARTHUR N. RAMOS
BSEd-3B Science
Culturing Microorganisms from Environmental

Samples
(Growth media)
General Objectives:
1. Learn how to inoculate growth media using proper aseptic procedures

2. Learn how to streak for single, double, full mount colonies.
3. Understand the Inoculation of selective and differential media
4. Test some common antimicrobial herbal plants locally found in our backyard using
positive and negative control agar well technique.
Materials Needed:
 1000 ml purified water  Hot plate
 Agar Nutrient  Hot plate
 Alcohol burner  Inoculating loop
 Antibiotics  Magnetic stirrer
 Beaker  Petri dish
 Broth culture sample  Plant extract
 Cotton buds  Stirring rod
 Durham tube  Syringe
 Filter paper  Top load
 Flasks  Tissue
 Graduated cylinder  Herbal Plant extract (Papaya Leaf)
Agar Plate Preparation

Agar is a mixture of polysaccharides derived from red algae, it is used as a solidification
agent mostly in microbiology as it has the standard to culture media. It can be melt in high
temperature yet solid in room temperature, contains no nutrient where microbes can use to
grow yet its surface is conducive for the proliferation of microbes.
a) Sterile all the laboratory apparatus that will be used and get contacted and preparing
agar mixture (Petri dish, magnetic stirrer, Durham tube, stirring rod, etc.) using dry
heat method, hence through oven exposure.
b) Prepare the laboratory area, set all the apparatus to be used, disinfect the working
place including yourself. Use proper protective equipment. And be sure to turn off
electricians to avoid air pressure that will potentially carrier of other microbes.

BSEd-3B Science
c) Place the filter paper above the top load balance and weigh 10 grams of nutrient agar
powder. After that, twist the filter paper creating a cone shape then put it in a flask.
d) Using graduated cylinder, measure 250 grams distilled water then gently pour it on
the flask with Agar Powder.
e) Using the stirring rod, softly mix the nutrient agar powder and distilled water until the
powder is completely dissolved.
f) Put the magnetic stirrer into the Erlenmeyer Flasks and put the Erlenmeyer flask
containing the nutrient agar mixture on a hot plate and set it to the desired
temperature and the stirring speed to sterilize and mix the mixture.

BSEd-3B Science
g) Continuously check the temperature to prevent it from getting over cooked. Once the
mixture has reached a boil and becomes clear in appearance, remove it from the heat
source and allow it to cool.
h) Prepare 5 petri dish in a disinfected table then pour the Agar mixture into it until 1/4
of the petri dish’s height is filled. Do it gently and surely with minimal opening to
prevent the mixture from getting contaminated.
i) Let the Agar plate be cooled in a safe area for inoculation.


BSEd-3B Science
Note: Disinfect yourself every after step to avoid contamination.
Inoculation of selective and differential media
Petri dish no. 1

Single streak Agar plate
Single streak Agar plate

Single Streaking for isolation takes advantage of the characteristics of agar and solid
media and allows us to physically separate individual bacterial cells so that they each can
grow up into discrete, isolated colonies. It gives us the opportunity to have a clearer and non-
chaotic view of how microbe colonies proliferate as microbes are instructed to grow intensely
in a single strand manner.
Procedure in preparing inoculating media through single streaking.

a) Prepare the broth culture, alcohol lamp, inoculating loop in your disinfected working area. Be
sure that the cotton buds tip is conducive for broth absorption. Be sure also to place the
alcohol cap beside the lit alcohol lamp for easier fire termination. Be sure to have a trash bin
beside you for quick trash disposal.
b) Remove the cap from tube. Do NOT put the cap of the tube down on the lab bench—
hold it in your hand.
c) Flame the lip of the tube.

BSEd-3B Science
d) Place sterile portion of inoculating loop into broth, then remove.

e) Flame the lip of the tube
f) Replace the cap.
g) Gently streak the surface of an agar plate with the inoculating loop. STREAK IT IN A
SINGLE STREAK METHOD. Avoid contacting the inoculating loop from any parts of
the petri dish except the desired streaking strand.
h) Sterilize the inoculating loop and the side of petri dish using alcohol lamp.
Conclusion:
It created a dragon liked shaped, the stroke was perpendicularly growth by microbe
colony creating a rib like structure. From the top, irregular shape, raised elevation, and
undulated margin is being observed. Meanwhile, the mid to end part is in irregular shape yet
umbonated elevation, and lobated margin. At the right upper side of the agar plate, I saw a
dotted circular colonies aligned. Perhaps it is my mistake upon streaking. I was so sure that it
is not touched by the inoculating loop when I do the streaking but the air molecules from my
movement carried some of the microbes and dropped into that certain area. I reflected then
that when you do a streaking it is not only being fast and gently that is encourage, you must
do it with lowest possible pressure of movements to avoid air to travel which may carry your
samples into the surface of the agar plate.
Petri dish no. 2

Double streak Agar plate

BSEd-3B Science
Double streak Agar plate

Double streak is a method to which you will be doing double strand in a different part
of the agar plate. This method is used in order to have to perspective of single strand. Doing
single strand makes sure that you have a clearer view of how microbes proliferate, in double
streak method, it allows you to compare and contrast two strands to get a wider view about
the characteristics of microbe growth. It is also a way of double checking the behavior of the
inhibited colonies.
Procedure in preparing inoculating media through double streaking

f) Replace the cap.
g) Gently streak the surface of an agar plate with the inoculating loop. STREAK IT IN A
DOUBLE STREAK MANNER. Avoid contacting the inoculating loop from any parts of
the petri dish except the desired streaking strand.
Conclusion:
The two strand done by streaking is still observed yet very populated unlike the “Single
streak” where colony growth are more intact and more organized. In this streak, microbes are more
chaotic, they grow everywhere, and they don’t observed one specific area to proliferate. Same
assumption of problem that is observed in single streak agar plate is applied with this streaking.
Hence Like what I latter said, we must be more soft in creating movements so that we don’t trigger air
movement which then potentially carrier of microbes. Even though I did it carefully to make double
strand as precise as possible, my movements trigger the air and thus spread molecules from the cotton
I used to swab.

BSEd-3B Science
Petri dish no. 3

Full mount Agar plate
Full mount Agar plate

This streak plate method is a rapid and simple technique of mechanically diluting a relatively
large concentration of microorganisms to a small, scattered population of microorganism. The goal is
to obtain isolated colonies on a large part of the agar surface, so that desired species can then be
brought into pure culture.
Procedure in preparing inoculating media through single streaking.

f) Replace the cap.

BSEd-3B Science
g) Gently streak the surface of an agar plate with the inoculating loop. STREAK IT IN ALL
OF THE SURFACE OF THE AGAR PLATE.
Conclusion:
The agar plate is homed to a circular and irregular shaped microorganism with convex and
concave elevation with an undulated margin. It was streaked full mount but there are some that are not
inhibited by microbes, perhaps it is due to my excessive disinfection or uneven streaking technique.
Petri dish no. 4

Agar plate with Positive and negative control
Over all view where left agar well is the Over all view illuminated by the light below, where left
positive control and right is negative control. agar well is the positive control and right is negative
control.
Positive control:
Generic name: Dicyrine
Scientific name: Dicycloverine hydrochloride
Usage: Anticholinergic, it is a drug that is used to block and inhibit the activity of the
neurotransmitter acetylcholine (ACh) at both central and peripheral nervous system
synapses.
Dicyclomine hydrochloride is an antispasmodic agent. The MIC of dicyclomine
against standard strains of Gram positive and Gram negative bacteria were

BSEd-3B Science
performed by NCCLS broth dilution technique. These drugs showed a rapid killing
action on Gram positive bacteria, Staphylococcus aureus NCTC 6571, 8530 and
several other reference strains. The killing effect against Gram negative bacteria,
Shigella boydii 8 NCTC 254/66 and Salmonella typhimurium NCTC 74 showed
that the drug was bacteriostatic with respect to these strains.
Negative control:
Bottled purified water (H2O) is used as a negative control as it doesn’t have
the nutrient that help the microbes to proliferate yet it is a potential media for it to
grow. Water basically contains nothing in it, since it is inert, it won't affect the
outcome of tests completed for science projects. As a control element, when
conducting multiple science projects or tests, water that is pure won't change the
results of the test.
Procedure in preparing inoculating media with papaya leaf extract.

a) Dissolve 15 mg of Dicycloverine hydrochloride in a 250 ml purified water. Set aside in a
clean and safe area. Prepare a clean purified water for negative control which will be use
later.
b) Prepare the broth culture, alcohol lamp, cotton buds in your disinfected working area. Be sure
that the cotton buds tip is conducive for broth absorption. Be sure also to place the alcohol
cap beside the lit alcohol lamp for easier fire termination. Be sure to have a trash bin beside
you for quick trash disposal.
c) Disinfect your hands and make two agar well parallel to each other with equal division
equivalent to a half of the circle per hole to the Agar plate as quick yet gentle as possible. Be
sure to dispose the Durham tube right after you used it.
d) Hold the tube and petri dish with your hand. Remove the cotton cap from tube. Do NOT
put the cap of the tube down on the lab bench—hold it in your hand.
e) Flame the lip of the tube.
f) Place sterile portion of cotton bud into broth, then remove.
g) Flame the lip of the tube
h) Replace the cotton cap.
i) Gently swab the surface of an agar plate with the cotton buds. Swab it full mount. The set
aside.
j) Settle all the used things into their proper places, get the agar plate and with the use of a
permanent marking pen, mark one of the agar well for positive control identification.
Disinfect your hands.
k) Proceed to dropping of negative and positive control, with the use of syringe, have one drop
of positive control into the agar well designated for positive control. Again, have one drop of
purified water into the other agar well which will serve as a negative control.
l) Don’t forget to disinfect your hands after.
Conclusion:
All of the plate are all get populated. No agar well became resistant to microbe. Both
positive and negative controls doesn’t show any resistance to the cultured media. Conclusion is hard

BSEd-3B Science
as positive control displays a weak resistance creating a foggy yet thin layer of microbe inhibition.
This thin layer are scatter circular in a dotted manner around the positive control well. There is a
reaction yet conclusion is vague. Negative control is expected as there are still a lot of microbe
growth in around the agar well. Perhaps, the bacteria that the positive control can kill is low to me,
or the ratio of dissolving the drug is error, or the water used is unclean, or maybe, I somehow failed
to practice proper storage and end up populating the diffused area and the shallow dotted colonies
are the evidence of this lapses. As a reflection, next time I will do such experiment, be mindful with
the ratio, the proper storage, or maybe my disinfection and sterilization practices must be improve.
Petri dish no. 5

(Papaya leaf extract in an agar well)
Close-up view of agar well, illuminated

by light below for clearer view.
Over all view
Herbal used:
Ilocano name: Papaya
Common name: Papaya, Papaw plant, pawpaw plant
English name: Papaya
Scientific name: Carica papaya
The papaya, papaw, or pawpaw is the plant species Carica papaya, one of the 21 accepted
species in the genus Carica of the family Caricaceae. It was first domesticated in Mesoamerica, within
modern-day southern Mexico and Central America. In 2020, India produced 43% of the world's
supply of papayas. Papaya is also a local plant of Philippines since our tropic climate is conducive for
its production.
Papaya leaf was very effective against P. aeruginosa growth, followed by P. acne and S.
aureus. The potent killing effect of fermented papaya leaf supernatant showed a potential use as

BSEd-3B Science
bioactive ingredient in skincare application to control pathogenic microbe infection. (Derived from
“Evidence of potent antibacterial effect of fermented papaya leaf against opportunistic skin
pathogenic microbes” by Mohd Danial, A., *Koh, S.P., Abdullah, R. and Azali, A. et.al)
Procedure in preparing inoculating media with papaya leaf extract.

 Sanitize your hands and prepare the papaya leaf to be extracted. Wash it with water to be
followed by alcohol to ensure sterilization. Damp it with a tissue to dry it out then set aside in
a safe and clean area for later use.
 Prepare the broth culture, alcohol lamp, cotton buds in your disinfected working area. Be sure
that the cotton buds tip is conducive for broth absorption. Be sure also to place the alcohol
cap beside the lit alcohol lamp for easier fire termination. Be sure to have a trash bin beside
you for quick trash disposal.
 Disinfect your hands and make a hole in the center of the Agar plate as quick yet gentle as
possible. Be sure to dispose the Durham tube right after you used it.
o
 Hold the tube and petri dish with your hand. Remove the cotton cap from tube. Do NOT
put the cap of the tube down on the lab bench—hold it in your hand.
 Flame the lip of the tube.
o
 Place sterile portion of cotton bud into broth, then remove.

BSEd-3B Science
o
 Flame the lip of the tube
o
 Replace the cotton cap.
 Gently swab the surface of an agar plate with the cotton buds. Swab it full mount. The set
aside.
o
 Settle all the used things into their proper places then disinfect your hands.

BSEd-3B Science
m) Proceed to papaya leaf extraction using your thumb and index finger.
n) When you already confident enough that you already had a drop of extract, open the petri dish
half and drop the extract into the agar well.
Conclusion:
All of the part of the swabbed agar plate are got populated, yet there is a visible change that
can be observed around the agar well where papaya extract is been dropped. It is foggy yet its
cloudiness is far thinner than the microbe colony that grew around it. With 7 mm irregular shaped
(9 mm minus the Durham tube diameter which is 2 mm) that has been restricted from the microbes, I
therefore conclude that papaya leaf extract indeed contain antimicrobial property. And it seems to be
coincidence that I have some microbes that papaya leaf extract can suspend. What I conclude to its
characteristic where the diffused area is not totally clean but got a shallow microbe growth is that I
have multiple microbes that grow in my broth culture, it’s just a matter of fact that there are only
certain microbe that can papaya leaf extract can kill.
<----------=====*=====--------->
NOTE:
 Due to same technique being conducted in Petri dish 1-5, pictures are only included in P5 for general reference.
 Due to quality distortion when being passed through messenger, the presented picture may be blurry than the actual
picture.
o I just borrow cell phone for documentation, I am not confident about the pixel of my own camera hence I need
to pass the data through social media 😊.

Culturing Microorganisms From Environmental Samples

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Culturing Microorganisms From Environmental Samples

Uploaded by

Copyright:

Available Formats

Cagayan State University - Lal-lo campus | College of Teacher Education

SCI 70 Microbiology and