Microbiology

Part 1: Aseptic Technique

Objective

1. To have a better understanding of plate technique

2. To observe the effect of handwashing on bacteria on thumb

Introduction

Streak plate method is a technique used in microbiology to isolate a pure strain from a single
species of microorganism, most often bacteria which are true for this experiment. A
microbiological culture can be grown so that the organism can be identified, studied or tested.

The inoculation loop needs to be sterilized first so that contamination of any kind can be
avoided and then it is dipped onto the petri dish containing the E. Coli The loop is then
spread across a quadrant of an agar plate containing the bacteria that has been sterilized. This
allows the solution of the bacteria to be introduced to a substrate which provides them
nutrients, allowing them to grow. The choice of which growth medium depends on the type
of microorganism being cultured, growth media are usually based on agar, a gelatinous
substance. The loop is then re-sterilized and dragged across the inoculated quadrant of the
streak plate. This is done in order to collect some bacteria on the loop. The loop is once again
sterilized and spread around another portion of the plate much like the previous times. Each
time the loop will gather fewer and fewer bacteria until it just gathers just one bacteria cell
that is capable of growing into a colony. After that, the streak plate is then incubated, in this
situation for an approximate of 18 hours. The incubator is used so that the culture can grow at
an optimum temperature. After 18 hours, it can be seen that individual cells are present on the
middle of the plate.

The hands are also parts of the human body that are in most contact with the outside world.
People use their hands for a variety of activities every day, from opening the door to flushing
to toilet by pressing down the handle so it is extremely easy to come in contact with different
microbes and to transfer them to other objects and to the living organisms around.
Handwashing is thought to be an effective way for preserving the transmission of pathogens.
Eventhough there are many products in the market proclaim to be able to kill the germs but it
is not conclusive enough to say that handwashing with soap or other cleaning products is
more effective at reducing bacteria contamination than say using water.

Apparatus and materials

Inoculating loop, Bunsen burner, sterile water, Escherichia coli, Staphylococcus aureus, plain
water, hand sanitizer, soap, agar plate, marker pen, petri dish, parafilm

Procedures

(a) Streak Plate Technique

1. The inoculating loop in the Bunsen burner was sterilized by putting the loop into the
flame until it was red hot. Then, it was allowed to cool for a while.
2. The isolated colony from the agar plate culture of E. coli was picked and spread on each
of them over the first quadrant on separate agar plate.
3. The agar plate with the lid was covered and the loop was also flamed.
4. The plate was turned and light streaked into the next quadrant without overlapping the
previous streak.
5. Steps 3 and 4 was repeated and streaked into the thired quadrant.
6. Each plate was sealed with parafilm
7. The plate was inverted and incubated at 37oC for 24 hours.

(b) Effect of handwashing on bacteria on thumb

1. 4 nutrient agar was obtained and was labelled
a) Control
b) Water
c) Hand sanitizer
d) Soap
2. Each agar plate was divided into 4 sections by drawing lines using a marker pen on the
back of the petri dish.
3. Aseptic technique was used and the thumb was gently pressed on the control agar plate.
4. Hands were washed with water and step 3 was repeated on the appropriate agar.
5. Step 4 was repeated using hand sanitizer and soap.
6. Each plate was sealed with parafilm.
7. The plates were inverted and incubated at 37oC for 24 hours.

Results

(a) Streak Plate Technique

(b) Effect of handwashing on bacteria on thumb

Discussion

(a) Streak Plate Technique

From the results above, it can be observed that the experiment was a result. What should have
occurred was that there would be streaks of pure culture across the plate. Unfortunately, this
did not occur possibly due to my carelessness when handing the inoculating loop is because I
gouged the agar. This means that there would only be a few cells left on the loop to inoculate
the rest of the slant surface. However, the picture showed otherwise as there is no growth
whatsoever so I believed it was also because I did not use the streak technique correctly when
trying to put the loop from the isolated colony.

(b) Effect of handwashing on bacteria on thumb

From the results, the culture in each quadrant has no difference number of bacteria growing
on it. Whether or not this is due to the material or just a personal error is unknown.
What was supposed to happen theoretically was that the one with the highest number of
culture is the quadrant labelled control. The ‘control’ quadrant is the section where the thumb
was planted on it just to act as a base to compare with the other quadrants.

After washing the thumb with water and having planted the thumb on the quadrant labelled
‘water’, it can been noticed that it has a bit less culture grown on that section compared to the
‘control’ quadrant. While water is capable of removing bacteria, this did not decrease much
might be because the water isn’t hot, which makes it unable to completely kill the bacteria
effectively.

It also seemed that the culture grown on the quadrant labelled ‘hand-sanitizer’ has a similar
number to the ‘control’ quadrant. A possible explanation to this might be because the hand
sanitizer used for this experiment is non-alcoholic, meaning that it does not work well on
germs such as gram-positive and gram-negative bacteria. This also might be bcause the germs
have already develop a resistance to the sanitizer in question.

Lastly, when using soap, it is supposed to have less bacteria than by washing with hands
seeing as there are gaps in-between the culture in the ‘soap’ quadrant. This is possible due to
the fact that the soap does not kill the bacteria per say but instead, rinse them away so that the
bacteria lose their grip on the hand.

Conclusion

It can be concluded that a pure culture can be grown from E.coli using the plate streak
technique. By streaking, a single colony of bacteria can be obtained and straking on agar
plate can also make it easy to identify the bacteria. It is also best to have precautions to have
any pitfalls a shown in the results as this would give inaccurate results.

The effect on handwashing was not much different among the samples. However, in actually
it is by using soap to clean one’s hand, it is determined to be the method to remove the
bacteria the most while not cleaning it even a bit will lead to a huge amount of bacteria on it.
Also, hand sanitizer doesn’t necessarily work to remove bacteria depending on the bacterias.
Water can remove bacteria as well, it is just not as effective as using soap.

References

1. http://www.ruf.rice.edu/~bioslabs/bios318/318manual.htm
2. https://www.cdc.gov/handwashing/show-me-the-science-hand-sanitizer.html

Part 2: Gram staining

Objective

1. To differantiate between gram-positive and gram-negative bacteria

2. To have a better understanding of Gram staining

Introduction
Gram staining is a very common technique used to differentiate two large groups of bacteria
based on their different cell wall structure. Most bacteria can be divided into two groups,
basing on the composition of the cell wall, which are:

i) Gram-positive bacteria. They have a thick layer of peptidoglycan in their cell wall,
which contains crystal violet that these cells are stained with. A good indicator that
the bacteria in-question is a gram-positive one is that the cell walls are stained
blue/purple when Gram stain is applied.
ii) Gram-negative bacteria. Their cell walls are a bit more complex as they have a thin
peptidoglycan layer and an outer membrane beyond the plasma membrane and the
space between the plasma membrane and the outer membrane is called the
periplasmic space. Its thinner peptidoglycan wall also does not contain crystal violet
after the decoloring process. Gam-negative cells will also be stained pink with the
Gram stain.

Gram staining has three processes, which are: staining with crystal violet, decolorization and
counterstaining with safanin. Because of the differences in the thickness of a peptidoglycan
layer in the cell membrane between Gram-positive and Gram-negative bacteria, the Gram-
positive cell retains crystal violet during the decolorization process while the crystal violet in
Gram-negative bacteria does not.

Apparatus and materials

Bunsen burner, light microscope, immersion oil, 95% ethanol, safranin, Crystal Violet,
inoculating loop, Escherichia coli, Staphylococcus aureus, slide, sterile water, Gram’s Iodine

Procedures

1. The sterile inoculating loop was used and 1 drop of sterile was added to the slide, with a
smear of (a) Escherichia coli and (b) Staphylococcus aureus
2. The air was dried and heat was fixed.
3. The smear with Crystal Violet was covered for 1 minute.
4. The slide with water was gently washed off.
5. Gram’s Iodine was added for 1 minute.
6. Water was washed.
7. 95% of ethanol was decolorized. Stop decolorizing with alcohol as soon as the purple
color had stopped leaching off the slide, then washed immediately with water.
8. The smear with Safranin was covered for 30 seconds.
9. Both the top and bottom of the slide was washed with water.
10. The slide was blotted.
11. The light microscope was used to view the smear up to 100X with immersion oil.
Results

Samples Observation Gram(+/-)

Escherichia coli Negative

Staphylococcus aureus Positive

Discussion

In this experiment, gram staining was performed to have a better understanding of the
mechanisms behind it. Before starting the experiment, aseptic techniques were used to
minimize contamination. Two smears of microorganisms, which are Escherichia coli and
Staphylococcus aureus are used as samples for this experiment to differentiate between gram-
positive bacteria and gram-negative bacteria. Crystal violet dyes are liquids that have
positively charged particles that helps them bind to negatively charged molecule like teichoic
acid at a cell wall of bacteria. This crystal violet dye can dissociate into cv+ and cv- ions
which are capable of penetrating deep into the cell wall of bacteria, interacting with the
negative component of the cell wall.

Based on the results, it is clear that when Gram stain is used on E.coli, the cell wall is stained
bright red, making it gram-negative bacteria. This is because the ethanol acts as a
decolorizing agent that when interacted with the lipid membrane, will cause E.coli to lose
their outer membrane, exposing the peptidoglycan.

On the other hand, the Staphylococcus aureus is a Gram-positive bacteria seeing that the cell
wall is stained purple. This is because the CVI complex is being retained within the cell wall,
making the bacteria as it is. Ethanol was not added excessively to avoid the cell wall from
breaking, which causes no stain to be observed. Staphylococcus aureus is also no stained
pink when safranin is used because the peptidoglycan layer already contains CVI complex.

Conclusion

From the experiment that has been conducted, it is concluded that Gram staining is a very
reliable method of distinguishing between a bacteria that is gram-positive to one that is gram-
negative. Precautions were taken such as not using excess alcohol during the decolorization
process so that the stained cell wall is visible and present when viewing with a microscope to
ensure that the experiment is as accurate as possible and to avoid any potential errors. After
this experiment, I also have a better understanding on the usage of Gram staining.

References

1. https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
2. http://www.academia.edu/9655093/LAB_REPORT_OF_MICROBIOLOGY_3