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Microbiology
ABSTRACT
The human body has billions of bacteria which constitutes the normal flora fighting against the
invading pathogens. It is tedious to isolate a particular type of bacteria from a clinical sample.
Streak plate technique is used to grow bacteria on a growth media surface so that individual
bacterial colonies are isolated and sampled. Isolated colonies indicate a clone of cells, being
derived from a single precursor cell. When the selected culture media is inoculated using a single
isolated colony, the resulting culture grows from that selected single clone. The modern streak
plate method has evolved from the efforts by Robert Koch and other microbiologists to obtain
pure culture of bacteria in order to study them. The dilution or isolation by streaking procedure
was originally developed by Loeffler and Gaffky in Koch's laboratory, which involves the
dilution of bacteria by systematically streaking them over the surface of the agar in a petri dish to
obtain isolated colonies which will subsequently grow into mass of cells, or isolated colonies. If
the agar surface grows microorganisms which are all the genetically same, the culture is then
clarity of the microscopic image.Stains and dyes are widely used in the scientific field to
by the Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a
differential staining technique that differentiates bacteria into two groups: gram-positives and
gram-negatives. The procedure is based on the ability of microorganisms to retain color of the
stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the
alcohol, losing the color of the primary stain, purple. Gram-positive bacteria are not
decolorized by alcohol and will remain as purple. After decolorization step, a counter stain is
OBJECTIVES
1. To apply streaking plate method and obtain isolated microbial colonies from an inoculum by
2. To observe bacterial growth by apply those 3 control (using hand sanitizer, water, soap )
1. Agar plates
2. Inoculating loop
3. Bunsen burner
4. 70% alcohol
5. Slide
6. E –coli
7. Staphylococcus aureus
9. Safaranin
1. The inoculating loop was sterilized in the Bunsen burner by putting the loop into the flame until it
2. An isolated colony were chose from the agar plate culture of (a) E. coli and (b) S. aureus and
were spread each of them over the first quadrant on separate agar plate.
3. The agar plate were covered with the lid and flamed the loop.
4. The plate were turned and lightly streaked into the next quadrant without overlapping the
previous streak.
5. Step 3 and 4 were repeated and streaked into the third quadrant.
a) control
b) water
c) hand sanitizer
d) soap
2. Each agar plate were divided into 4 sections by drawing line using a marker pen on the back of
4. Wash hands (including thumb) with water and repeated step 3 on the appropriate agar.
1. Used a sterile inoculating loop, 1 drop of sterile water were added to the slide. A smear of:
3. The smear were covered with Crystal Violet (primary stain) for 1 min.
7. Decolorized with 95% ethanol. This is the "tricky" step. Decolorizing with alcohol has to stop
as soon as the purple color has stopped leaching off the slide (time will vary depending on
thickness of smear). Immediately wash with water. Be sure to dispose of all ethanol waste in the
9. Both the top & the bottom of the slide were washed with water.
11. Using the light microscope, the smear were viewed up to 100x with immersion oil.
RESULT
Laboratory assistant prepared us with this two different types of bacteria. I chose E-Coli.
Streaking plate method applied onto the plate. This is after the streaking take place. Before the
incubation phase
This is my plate before the observation with the 4 control.
Streaking plate method is a common method used to isolate certain colony of bacteria and
culture it in the agar plate with enough nutrients needed for the bacterial growth. The agar plate
contains various of nutrient needed for the bacteria to support it rapid growing. Streaking plate
method also use as an dilution method of bacteria. In this experiment we used pure culture of E-
Coli and dilute it onto other agar plate. The result of the experiment as the picture shown from
the above page. During the 4th streaking on the 4th quadrant were observed to have few colonies
of E-coli bacteria. Rather than first quadrant shown that, rapid growth of bacteria taking place.
Bacteria display a wide variety of nutritional and physical requirements for their growth.
This includes water, a source of energy, sources of carbon, sulfur, nitrogen phosphorus, minerals
such as Ca2+, Mg2+, Na+, and other vitamins and growth factors. Nutrient agar is a complex
medium as it contains ingredients with unknown amounts or types of nutrients. Nutrient agar
typically contains 0.5 % peptone, 0.3 % beef extract, 1.5 % agar in water (pH adjusted to neutral
at 25 °C).
For the next part is, observe the bacterial growth on the agar plate using 4 control stated.
For my plate, it resulting almost no change for bacteria growth in each quadrant. It showed that,
the same growth pattern for the 4 control in each quadrant. I do make a conclusion that, maybe
during the preparation of this control, my techniques applied is wrong from it original procedure.
We do know that microbiology experiment is so sensitive, as it involves with microbes, and our
experiment maybe contaminated with the several of microbes from air, from the table or
anywhere else. Speaking was not allowed and microbes form our clothes may be affected our
result to. This is all factors that could contribute to the failure result. But I do learn during this
The Gram stain is a very important preliminary step in the initial characterization and
staining characteristics, enabling the bacteria to be examined using a light microscope. The
bacteria present in an unstained smear are invisible when viewed using a light microscope. Once
stained, the morphology and arrangement of the bacteria may be observed as well.
The Gram stain procedure enables bacteria to retain color of the stains, based on the differences
in the chemical and physical properties of the cell wall. Gram positive bacteria can stain dark
purple due to retaining the primary dye called Crystal Violet in the cell wall. Staphylococcus
aureus is an example of gram positive bacteria while Escherichia coli is a gram negative
bacteria. Gram negative bacteria can stain red or pink due to retaining the counter staining dye
I learned so many things during the streaking plate method which is quite sensitive to
handle as we did not want those microbes in the air to affected our experiment.Only E-Coli
needed! We may have different colonies of bacteria if those streaking method applied is wrong.
And everything started with sterilization and sterilization. Sterilization is sterilizing media to
above 1210C for 15 minutes in an autoclave destroys nearly all living cells and spores. If screw
caped bottles are used, the cap must be loosened prior to sterilization process. So before we start
the streaking techniques, we must rub all those material and apparatus needed with alcohol and
I also failed handling the procedures using those 4 control to observe bacteria growth. We
should get different result but maybe the way conducting the experiment were wrong,
During the gram staining process I learned how to handle the apparatus very well. It is
not easy at first for us as this technique is new to us but as soon demonstrated, I feel excited to
try.Using the microscope to observe quite difficult without the actual procedures. I learned from
my friend and lecturer for the trick using microscope. The key is, patient and keep playing with
the knob.
REFERENCES
https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1
http://vlab.amrita.edu/?sub=3&brch=73&sim=213&cnt=1