Polytechnic University of the Philippines

Sta. Mesa Manila

College of Science
Department of Biology

BIOL 30035
General Microbiology Lab
Laboratory Activity no. 2

Aseptic technique and smear preparation: Journey in eliminating the odds

of contaminating the colony and utilizing every resources
Amada, Gabrielle Kyle D., Amigo, Justin M., Bahillo, Angelo James A.,
Diaz, Gavin Vance Ric M., Jaramillo, Arnold Daniel B.

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Abstract:
This lab activity presented the various equipment and techniques that is
required for the study of microbiology. With the fundamental principle of aseptic techniques
which practice to prevent contamination from harmful pathogens in order to minimize the
risk of infection. Each student perform different techniques from flaming the mouth of a test
tube to pouring of agar in the petri dish closely near in flammable alcohol lamp for
decreasing of chances of contamination. Different streak of inoculating loop is also
performed with has three ways to smear the simple streaking, the t streaking, and the 4
quadrant streak. Streaking techniques are used to isolate pure stains from single species of
microorganisms. This study concludes the importance of proper use of equipment and
practice of proper procedures for hygienic and safety purposes of every individual in
laboratory.

Keywords: Agar, Aseptic technique, Streaking techniques

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Contributions:
Abstract/ Keywords/Title: Amada
Introduction Bahillo
Methodology: Diaz
Results and Discussion: Amigo
Conclusion/References: Jaramillo
 I. Introduction
Microbiology is a branch of science that deals with microscopic organisms such as
bacteria, viruses, archaea, fungi, prions and protozoa collectively known as “microbes” and
their individual activities. These organisms play vital roles in the cycling of nutrients,
biodegradation, and climate change, spoilage of food, viral diseases and biotechnology. Due
to their natural structure, they are being cultured and used by humans in ways like; drugs and
vaccines to different infections, manufacturing of biofuels, getting rid of pollution, food
processing and more. (na, 2019)
In studying this field of Science, several techniques that require specific time and
conditions are needed to be practiced, primarily in handling and culturing the specimens.
Glass wares, laboratory equipment and PPE (personal protective equipment) are standard in
performing experiments.
Glass is used in laboratories because of its versatility to handle microbes and
chemicals. Glass can be blown, bent, cut, molded, can be transformed into different sizes and
shapes and the ability to be re-used again, of course with proper re-sterilization. (Zhang,
1999)
Certain species of microbes may bring infections to the ones who handle it, so after
familiarizing glass wares and laboratory apparatus, scientists are required to memorize ways
of preventing contamination and use of proper & pure culture preparations.
Another fundamental principle in handling microbes, is knowing how to practice
aseptic techniques which are practices and procedures that prevent contamination from
harmful pathogens which involves applying even the strictest rules in order to minimize the
risk of infection. (Cherney et., al, 2017) It prevents cultures, media stocks, and other solutions
from being contaminated by these harmful microbes. Smear preparation is another basic
practice needed in microbiology. Smear is a sample which is spread on a slide to be observed
in a microscope or in a surface of another medium to be cultured. It also prevents samples
from being lost in a staining procedure. It is prepared in a solid or broth medium. (na, nd)

This laboratory activity aims to provide a basic overview on the uses and instruction
for the usage of different laboratory glass wares and apparatus that are being used in a
laboratory. It also aims to acquire the skill of aseptic techniques in the microbiological field
and let the scientists practice it to prevent contamination of cultures and media from microbes

Contributions:
Abstract/ Keywords/Title: Amada
Introduction Bahillo
Methodology: Diaz
Results and Discussion: Amigo
Conclusion/References: Jaramillo
in the environment. Lastly, another purpose of this activity is to give basic knowledge on how
to perform basic smear preparations which are always used in Microbiology.

II. Methodology

The materials used in the activity were a packet of powedered gelatin, lukewarm water, a
large bowl, a stirring rod, 2 test tubes, an alcohol lamp, an inoculating loop, an inoculating
needle, 5 petri dishes, and methylene blue.
Different laboratory apparatus were illustrated and labeled in according to their function.
These different laboratory apparatus include the inoculating loop, the inoculating needle,
glass slides & cover slips, test tubes, the test tube rack, petri dishes, the stirring rod, the
pipette, the dropper, beakers, graduated cylinders, erlenmeyer flasks, candle jars, aspirators,
wash bottles, autoclaves, incubators, electric ovens, alcohol lamps, the spatula, applicator
sticks, laminar flow hoods, concave slides, and the weighing balance.
For the gelatin preparation, 112.5 ml of lukewarm water was poured into the large bowl. 1
packet of powdered gelatin was then poured into the water as evenly as possible in terms of
distribution. The group waited 5-10 minutes for the gelatin to finish blooming (expanding). 1
½ cups of water were then heated over medium heat until it began to simmer. The hot,
recently-simmered water was then poured on the gelatin. The gelatin was then stirred using
the stirring rod until it dissolved in the water. The gelatin was slightly cooled down before it
was poured into the petri dishes where it would solidify.
A quarter of a test tube was then filled with the gelatin. The test tube was layed at an angle
that made the gelatin have a slanted surface as it cooled down and solidified.
For loop flaming which sterilized the inoculating loop, the group then performed the
following:

The tip of the inoculating loop was held in the flame of an alcohol lamp until it
glowed red-hot for a few seconds. After flaming, the inoculating loop was slightly cooled
down.

After the loop flaming, the steps for simple streaking were then performed. Methylene
blue was put into a test tube. The mouth of the test tube was flamed before the inoculating

Contributions:
Abstract/ Keywords/Title: Amada
Introduction Bahillo
Methodology: Diaz
Results and Discussion: Amigo
Conclusion/References: Jaramillo
loop was dipped into the methylene blue. Before lifting the lid of the petri dish in order to
insert the inoculating loop, the edge of the petri dish cover was flame-sterilized. The
inoculating loop was then lightly dragged in a zigzag formation from the top to the the
bottom of the gelatin’s surface without damaging the gelatin itself. After dragging the
inoculating loop, a zigzag from/S-formation was visible in the smear formation. The opened
part of the petri dish was flame-sterilized before closing. The inoculating loop was also
flame-sterilized. The loop flaming and simple streaking steps were performed separately for
all 5 petri dishes with a member of the group performing the steps with a petri dish assigned
to each of them.

III. Results and Discussion

Fig1. Preparation of agar (gelatin), specifically flame- sterilizing

the petri dishes.

Contributions:
Abstract/ Keywords/Title: Amada
Introduction Bahillo
Methodology: Diaz
Results and Discussion: Amigo
Conclusion/References: Jaramillo
 First thing in preparation of agar, you need to pour 112.5 mL of water into a bowl,
and then you need a pack of gelatin; only 1 tablespoon is needed. Upon pouring the gelatin,
stir it; wait for 5-10 mins for gelatin to bloom. “Blooming” is an occurrence wherein the
gelatin absorbs the liquid to ensure a smooth texture of finish product. Immediately, add the
simmered water (337.5 mL) into the bowl, careful not to use boiling as it will destroy the
gelatin. Keep stirring until there are no visible granules.

For the aseptic technique proper, given that above steps are already satisfied; first, we
need to flame the mouth of a test tube and then cover it, cotton was used in this instance. The
main purpose for such (flaming) is to create air convection current, this makes air rise up that
helps to prevent dust that might contaminate the test tube. Upon securing the test tube, ready
your petri dish for agar to be transferred. First step, put the cotton cover (test tube) in between
your ring and pinky finger, make sure that you wouldn’t touch any surface, then flame the
mouth of the test tube, same reason as earlier, at that instant, flame the sides of the petri dish
to avoid contamination from other agents. Next, slightly open the petri dish and then pour the
agar from the test tube; take note, don’t open widely the petri dish because if you do, you’re
increasing the chances of making it contaminated and you must work closely to the flame so
you’re constantly making air convection currents. Immediately, flame the mouth of the test
tube and return the cover. Finally, flame again the sides of the petri dish right after pouring
and securing the test tube.

The very purpose of this experiment is to practice the proper ways to perform aseptic
technique; moreover, aseptic technique is used to maximize the absence of different
microorganism such as pathogens that might cause diseases, by that, we can successfully
culture different organisms and perform the experiment as we want in a laboratory setting.

The second activity was all about different ways of streaking; it is a technique in
isolating a colony of microorganisms in order to study the morphology of that specific
colony, it can be done by the means of spreading a dipped inoculating loop (methylene blue)
into the agar plate (petri dish). There are different types of streaking, and those are simple, “t”
and, four quadrants. Furthermore, this method was introduced to help in transferring from
large quantity of microorganism into smaller concentration, when the organisms are taken it
can now be identified, studied and, teste
Contributions:
Abstract/ Keywords/Title: Amada
Introduction Bahillo
Methodology: Diaz
Results and Discussion: Amigo
Conclusion/References: Jaramillo
Fig2. Simple streaking method Fig3. “T” streaking method
Source:
Source: http://www.fao.org/3/ab768e/AB768E02.htm http://physionet.cps.unizar.es/tutorials/epn/html/chp51
exp2.htm

Fig4. Four quadrant streaking method

Source: https://microbeonline.com/streak-plate-method-
principle-purpose-procedure-results/

Fig 2, 3, 4 shows few of the different streaking techniques, namely, simple, “T” and,
four quadrants. Three and four quadrants are commonly used inside the laboratory and
clinical setting; the first streak is where most of the growth can be found and then density
decreases and younger organisms where found in the latter streaks.

Contributions:
Abstract/ Keywords/Title: Amada
Introduction Bahillo
Methodology: Diaz
Results and Discussion: Amigo
Conclusion/References: Jaramillo
 Table 1. Different laboratory equipment and their functions.

Equipment Function

Inoculating loop Pick up small sample from tube

Inoculating needle Transfer and inoculate living microorganism

Glass Slides & cover slip

Holds the specimen and protects before observation

Test tube
Storing, culturing and, mixing of microorganisms
Test tube rack Holds and stores test tubes
Petri dish
Culturing different microorganisms
Stirring rod Mixing different chemicals
Pipette Transport of measured solvents
Dropper Transfer solvents
Beaker Storage and mixing
Graduated cylinder Measuring solvents
Erlenmeyer flask Mixing different chemicals
Candle jar Culturing anaerobic microorganism
Aspirator Removes gases and liquids
Wash bottle Portable tap, for cleaning different areas
Autoclave Sterilizing different apparatus
Incubator
Grows and maintain microbiological cultures

Electric oven
Heating or attaining a certain temperature, drying and sterilizing
Alcohol Lamp Heat source, flame sterilizing
Spatula Scrapping, transfer of microorganisms
Applicator stick Obtaining and transferring samples
Laminar flow hood Prevents contamination of cultures of organisms
Concave slide Holds mostly big organisms or liquid to avoid overflowing
Weighing balance
Measuring different specimen or organisms
Contributions:
Abstract/ Keywords/Title: Amada
Introduction Bahillo
Methodology: Diaz
Results and Discussion: Amigo
Conclusion/References: Jaramillo
 Table 1 shows different necessary laboratory equipment needed in microbiology;
these tools will help you to do a lot of observations and experimentations regarding different
microorganisms. Specifically in this matter, these tools will help you in growing or culturing
your microorganisms as well as studying and identifying such. Moreover, you need every
other equipment in order to perform a successful experiment; for instance you need an auto
clave to sterilize your other tools, thus, every equipment is vital for the observations and
experimentations.

Guide Questions:
1. What is the purpose of knowing the functions of the equipment/apparatus necessary in
Microbiology?

 Safety, it’s important that we’re knowledgeable about the proper lab
equipment because wrong usage might lead into accidents. Efficiency, if we
use the proper equipment, we can utilize or maximize our limited resources.

2. Why do you need to know proper aseptic techniques?

 Aseptic technique is really vital in microbiology lab as we deals with sensitive

organisms; thus, this technique helps us to prevent possible contaminations
that might affect the results of the experiment.

3. How much time is required to properly sterilize inoculating loops and needles?

 Wait until your inoculating loops or needles to become red/orange hot before
using it.

4. What is a CFU?

 Colony-forming unit, used for estimating the total number of microorganism

present in your culture.

5. What is a pure culture?

 A pure culture is where only one species of microorganism is present, it’s also
called as axenic

6. What is a colony?

 It’s a visible mass of microorganism present in your culture.

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 7. How does streaking technique works?
 Streaking works by isolating a certain pure stain of microorganism and
transferring it into another medium by the means of spreading it into the agar
plate, in this case gelatin was used.

IV. Conclusion

This lab activity presented the various equipment and techniques that is required for
the study of microbiology. There are a lot of tools and equipment used in microbiology such
as the test tubes and flasks that are used to store liquids, Petri dishes and incubator used to
culture samples. There are also tools used for sterilization like the alcohol lamp and the
autoclave that kills microorganisms by heat. Equipment are important in microbiology
because there are some things that cannot be done by human hands alone and this is why
these tools are important.

For the activity the students are taught the various techniques required in
microbiology and that is the aseptic techniques and smearing. Aseptic technique is important
because these are practices and procedures that prevent contamination from pathogens which
may lead to sickness. Aseptic techniques are also used in surgery for hygienic and safety
purposes. In this lab activity the first thing done is preparing the agar in the petri dish, this
will serve as a food source for the microorganisms. The agar is first cooked by adding
simmered water to the gelatin mixture. Don't use boiling water as this will ruin the gelatin.
Next is flaming the mouth of the test tube and petri dishes, these will prevent microorganisms
from starting to grow. The next step is flaming the inoculating loop which will sterilize it.
After these there are three ways to smear; the first one is the simple streaking, the next one is
t streaking, and the last one it the 4 quadrant streak. Streaking techniques are used to isolate
pure stains from single species of microorganisms.

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 V. References
Cherney et. al, 2017 “Aseptic Technique” Retrieved from:

https://www.healthline.com/health/aseptic-technique

[Smear Preparation] (n.d.) Retrieved from: https://windward.hawaii.edu/facstaff/miliefsky-

m/BIOL%20171L/Lab%2013%20Prokaryotes/gramstainprocedure.pdf

What is Microbiology. 2019 Retrieved from: https://microbiologysociety.org/why-

microbiology-matters/what-is-microbiology.html

Zhang, 1999 . "Laboratory glassware as a contaminant in silicate analysis of natural water

samples". Water Research.

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