Palestine Polytechnic University

Faculty of Medicine & Health Sciences

Medical Microbiology Laboratory
3rd Experiment
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Cultivation Methods & Sterilization

Streaking Blood Agar & Tryptone Glucose Yeast Extract Agar

04 October 2021

Written by the student: The supervisors:

Mohammad Fadel Khaleel Nu`man Dr. Aya Ghanaym
190007 Dr. Rawan Bseileh
Class: 3 .. Monday (12:00 – 02:00)
Procedure of streaking blood agar experiment:
1_ First of all, the whole tools used in this experiment should be put in the safety cabinet, also working should
take place there to avoid any probable infection. The blood agar media that was prepared in the last week`s
experiment will be used in this laboratory.

2_ Using an inoculation loop, take an isolated colony from the broth culture which contains E. coli bacteria,
and then spread the colony over the 1st quadrant of the plate gently, by following back and forth motion in
close parallel streaks.
3_ By using other loop, repeat the same previous motion from the edge of the first area, to extend the streaks
into the second quarter of the plate. Repeat this step in the third and the fourth quarters till the colony is
extended to the four quadrants, one by one.

4_ At last, the plate is put in the incubation for 24 hours at 37℃.

Discussion:
It is important to dilute the colony inoculated by streaking it on
the plate surface till we have only one cell of the bacteria
deposited every few millimeters on the surface of the agar plate.
After some time, these single cells proliferate and become
thousands of new bacterial cells, which is referred to as "isolated
colony", and this method is followed to get a pure culture. After
24 hours of waiting, all colonies should be having the same
appearance in general, unless if contamination has happened
during the procedure.

In the following plate, it is clear that the first quadrant came out
with a huge growth, which is gradually descending in amount in
the second, third, and fourth quadrants

Procedure of noticing the effect of disinfectants on bacteria experiment:

1_ Label the TGY (Tryptone Glucose Yeast Extract Agar) media dish into four areas.
2_ Using distilled water, dilute the sample of bacteria (taken from an isolated culture).
3_ Swap the bacteria by cotton swap into the TGY media dish before extending it. Make sure that lines made
must be compact without spaces and looking like a carpet.
4_ Four filter papers are added to the TGY media dish, each in an area that were made in step 1.
 The first filter paper was put previously in a plate of Dettol, and then in area 1.
 The second filter paper was put previously in a plate of 70% alcohol, and then in area 2.
 The third filter paper was put previously in a plate of 95% alcohol, and then in area 3.
 The fourth filter paper was put previously in a plate of chlorine, and then in area 4.
Discussion:
Waiting for 24 hours of incubation will show the effect of disinfectants.
If the disinfectant used is the strongest against this type of bacteria, it
will cause huge clear domain around the filter paper. In our experiment,
it was clear that the chlorine was the strongest disinfectant.

Alcohol Alcohol Chlorine

Bacteria Media Dettol
(70%) (95%) (3%)
E. coli TGY +ve +ve +ve +ve
S. aureus TGY -ve +ve +ve +ve
Questions:
1_ Which disinfectant was the best according to your results? How was the effect of the same
disinfectant on different bacteria? Give explanation for your answer?
In our experiment, the best disinfectant was the chlorine for both of the bacteria, as the clear area around the
filter paper was the largest when the paper was put in a chlorine sample, which means that the chlorine has
killed the highest number of bacteria when compared with other disinfectants.
The chlorine`s effect on S. aureus (gram-positive) was greater than its effect on E. coli (gram-negative), since
gram-negative bacteria have higher resistance to disinfectants 1.

2_ In theory 70% alcohol is more efficient than 95% alcohol as an antiseptic or disinfectant,
why? Did you get such a result, if not, Why?
The usage of the more concentrated solutions (99%) will result in almost immediate coagulation of surface
or cell wall proteins and prevent passage of the alcohol into the cell. A 70% isopropyl alcohol can
effectiveness cross over the cell membrane, thereby attacking the entire cell and killing the bacteria (CDC, 2020).
The benefits of using 70% alcohol are:
1_ Coagulation of surface proteins proceeds at a slower pace, thereby allowing the alcohol to enter the
cell.
2_ 70% alcohol, being a dilution of absolute alcohol, contains water which is essential in the denaturing
process of proteins.
3_ Due to the concentration difference of water and alcohol on either side of the cell wall, 70% alcohol
enters the cell to denature both enzymatic and structural proteins. This increases the potency of its
antimicrobial properties.
And that is typical to what we had as a result in the lab.

3_ What is the objective of streaking microbial cultures over a broad agar surface?
The application of microorganisms to the medium surface within a plate, and the spreading of those
organisms with a loop, needle or glass rod is called streaking, and the resulting preparation is called a streak
plate. The objective of this technique is to dilute the culture, and to produce well-isolated colonies from a
concentrated mass or suspension of cells. Streaking over a broad agar surface provides a technique by which
mixed cultures can be separated since colonies of different types can usually be isolated by this means2.

4_ What is a pure culture and what method is most commonly used to obtain a pure culture?
A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other
species or types3. Streak plate method is used most commonly to isolate pure cultures of bacteria. A small
amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface
of the agar medium. The successive streaks “thin out” the inoculum sufficiently and the micro-organisms are
separated from each other4.

1 Bioscience Horizons: The International Journal of Student Research, Volume 10, 2017,
hzx008, https://doi.org/10.1093/biohorizons/hzx008, Published at 28 July 2017
2
Bykowski, T., & Stevenson, B. (2008). Aseptic Technique. Current Protocols Essential Laboratory Techniques, 00(1).
https://doi.org/10.1002/9780470089941.et0401s00
3
Libretexts. (2021, January 3). 6.4B: Pure Culture. Biology LibreTexts.
https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/6%3A_Culturing_Microorg
anisms/6.4%3A_Microbial_Culture_Methods/6.4B%3A_Pure_Culture
4
Garg, M. (2016, September 16). Obtaining Pure Culture of Microorganisms: 6 Methods. Biology Discussion.
https://www.biologydiscussion.com/organism/culture-organism/obtaining-pure-culture-of-microorganisms-6-
methods/55042