WEEK 6 ATTACHMENT SHARING

Hey guys, Johanna here. I am currently working in a research centre that deals with a variety of bacterial work. My research project focuses on protein work of Stenotrophomonas Maltophilia, a bacteria that has been known to cause noscomial infections in hospitals.

My Attachment

My SIP supervisor required us to perform a series of experiments to get a feel of how to run certain methods of bacterial identification tests based on the proteins we have extracted from the bacteria.

Our first SIP assignment was to perform Chloroform Shock on the bacteria. Choloroform shock is a method of extracting the cell surface membrane proteins that would most probably have cell adhesion properties. In the clinical context, this would probably mean that the bacteria's cell surface proteins actually help in adhering to surfaces like hospital equipment, furniture, patient's skin etc. Thus by extracting these proteins, we can carry out studies on these proteins to determine their clinical significance and also provide a basis for further scientific studies on how to fight these rapidly spreading microbes in hospitals.

The chloroform shock method is suppose to be specific to the extraction of cell surface membrane proteins. My group was suppose to determine if it is actually specific and does not cause cell lysis. Cell lysis would affect the experiment because the intracellular proteins would also be released, thus the proteins would not be purely cell surface membrane proteins. We ran the experiment about 6 times before we succeeded in proving that when using chloroform shock, mainly cell surface membrane proteins were extracted. Although there was cell lysis, it was not substantial enough to affect the results of the main experiment.

Our second SIP assignment was to standardize the protocol for the extraction of secretory proteins of the bacteria, S. Maltophilia. The secretory proteins are also clinically significant as these proteins would facilitate in the survival of these bacteria. This is because, different proteins should be secreted in the different clinical environments mainly at different temperatures. However, our SIP supervisor gave us two different clinical isolates, mainly the clinical isolate and environmental isolate. We were suppose to meet a series of objectives - to standardize the inoculum, to determine the cell density of the bacteria after 24hrs of growth and to correlate between the cell density with the number of cells. I will elaborate more on this experiment.

Before performing each experiment, we were suppose to plan out our protocol such that we would not collide with other people's experiments. Also, by planning our protocol, we would be sure of our next step. An example of our protocol would be:

10 July 2007 - Streaking

11 July 2007 - Inoculation

12 July 2007 - Cell density, Serial Dilution and Plating
(i) Methods
a) 18 agar plates were incubated in 37 incubator to dry for subsequent use
b) Centrifuge was set pre-cool and BSC was turned on
c) Materials required were swabbed and placed in BSC (Biosafety Cabinet)
d) Eppendorf tubes and agar plates were labelled
e) Tubes from 28 incubator were taken out, tapes removed and caps tightened
f) Tubes were centrifuges at 3,000xg for 20min at 10 degree Celcius
g) Supernatant was cecanted and cell pellet was resuspended in 10mL of LB broth
h) Washing step was repeated twice
i) Supernatant was decanted and cell pellet was resuspended in 20mL of LB broth
j) 1mL of LB broth was aliquoted into a disposable cuvette to tare cell density meter
k) 1mL of sample is then aliquoted into dosposable cuvette and measured
l) Each sample was measured in triplicates
m) Serial dilution was performed for chosen samples
n) The 6th, 7th and 8th dilution samples were plated on LB agar plates in triplicates

After performing those steps, we will incubate the plates for 24 hours before doing a plate count. from the plate count, we can determine if our dilution was performed correctly. If we did it right, there would be an obvious dilutional effect. Then, we can compare the results from the cell density readings and plate count to determine the number of cells in 1 OD (optical density).

When we carried out this experiment the first 4 times, we had a series of contamination cases. We carried out a lot of troubleshooting for the first two times and found out that the micropipette we used was contaminated. The following two tries, we still had contamintaion though it was significantly lesser than the first two and our results were not badly affected. However, to collect solid evidence that the protocol would provide good data, we were suppose to carry out more tests to confirm this.

Reflecting

Besides all the experiments we carried out, we also had to prepare all the materials that we require in the lab for our experiments such as the LB broth, LB agar, PBS used for serial dilution, DI water etc. We also had to keep the labs clean to enable easy assess to equipments and such. We also had to read a lot of protocols and literature reviews on the machinery we were to use such as the Xcise, MALDI TOF/TOF and the soon to come laser scanner for CyDyes.

Therefore, being in a research laboratory really teaches us a lot and makes us think on our feet and how to handle problems by troubleshooting and solving the problem. It also helps us to organize our time and think about others that work in the lab.

That's all i have to say for this post. Any questions don't hesitate to ask OK? See you guys in school soon!!

Johanna
TG02