WEEK 9

WOWWWW. Time flies. It's the 9th week =)) Hope u guys are coping well.

Anyway, this week, i will be talking about yet another skill that i picked up during my SIP - IMMUNOFLUORESCENCE. I believe just last week, Ye Tun has talked about this technique (http://sevenseven77.blogspot.com/) that he is using for viral identification. In my case, I am using it to show the localisation of a particular protein in the cell (whether it's in the nucleus or cytoplasm). It 's something really similar to Immunohistochemistry which i mentioned in WEEK 3 so i will skip explaining several steps ok? =)

Aim of test: I am using TWO primary Ab and of course 2 secondary Ab labelled with fluorescence substrate. The 2 fluorescence substrate i am using are FITC (giving the apple green fluorescence signal) and the TRITC (giving the red fluorescence signal) on one single section. Hence, when viewed under the fluorescence microscope, you should be able to see both colors!

Step 1 - 10 : Pls refer to the immunohistochemistry steps i wrote for WEEK 3

Step 11: Secondary Ab labelled with FITC was added. Since this 1st primary Ab that i am using is raised in mouse, my secondary Ab is an anti-mouse solution yup! Addition of Secondary Ab must be done in the dark so as to prevent the exposure of the fluorescence substrate and the slides was placed in a dish and wrapped in aluminium foil for the same purpose.

Step 12: Washing carried out

Step 13: This time round, the 2nd different kind of primary Ab was added

Step 14: Secondary Ab labelled with TRITC was added. Since this 2nd primary Ab that i am using is raised in rabbit, my secondary Ab is an anti-rabbit solution yup! Addition of Secondary Ab must be done in the dark so as to prevent the exposure of the fluorescence substrate and the slides was placed in a dish and wrapped in aluminium foil for the same purpose.

Significance: It is very important that you choose the primary Abs raised in different animals should you be using 2 antibodies on one single slide. This is because if both my 1st and 2nd primary Abs are raised in mouse, when secondary Ab is added, it will bind to BOTH the primary Abs instead of just one. Then, when you obtain your results, you wouldn't know if the Secondary Ab is binding specifically to its specific primary Ab.

Step 15: Washing was carried out once again.

Step 16: DAPI was added as a counter stain

Significance: DAPI is able to allow the nucleus of the cells to emit blue fluorescence signal. Hence, when viewed under the microscope, we would be able to know which cells are not emitting the fluoroscence at all (by only showing the blue DAPI fluorescence without the red/green fluorescence signal), hence showing no expression of that particular protein of interest.

Step 17: Washing done again

Step 18: Mounting medium was added onto the slide and cover-slipped.

Step 19: View under the fluorescence microscope! =)

As you can see from this pic, there are both the green and red fluorescence. In area where there is a slight tinge of yellow/orange, it is most likely where the 2 different proteins (Remember we are using 2 primary Abs?) co-localize. However, we need the confocal microscope in order to confirm if these 2 proteins are located in the exact same area.

Hope u guys understand my entry. haha. n take care =))

Chen Kangting
0503331A
TG02

Week 8 Attachment Sharing

Hey wait! Don’t switch to another blog. One of my colleagues gave me a unique and interesting exam question given in a university. If one day you become a lecturer, you should set such questions. Go read the 1st comment for this post!

Ok..now the boring part..

This few weeks I have learnt quite a lot of new stuff like flow cytometry analysis, plasmid purification, gel retardation assay and also confocal fluorescence microscope(still learning). However, I will focus on flow cytometry in this week post because I think it is more interesting.

Introduction

In this experiment, cancer cells are tranfected with polymer-DNA complexes. Since naked DNA/plasmid cannot be efficiently delivered into the nucleus of the cells, my lab designs carriers such as cationic polymers to deliver the DNA into the cells. In this case, the DNA is an EGFP encoded plasmid, meaning, cells that receive the plasmid are able to express the green fluorescence protein(GFP). Such DNAs are also termed reporter genes. To measure how good the carriers are in delivering the plasmid, we measure the level of gene expression.

Purpose of flow cytometry in this experiment

Flow cytometer is used to measure the gene expression. Cells tranfected with the EGFP plasmid will fluoresce green when exposed to blue light. The unique factor of flow cytometer is that it can measure the fluorescence per cell, hence you can know how many cells are expressing the gene. Unlike normal spectrophotometers which measure the transmission of light in a bulk of sample.

Flow cytometers can measure more that 1 type of fluorescence simultaneously per cell and also measure different types of cell. However in this experiment, the cell type used is HEK 293(human embryonic kidney cells) and the fluorochrome is green fluorescence protein(GFP). The brand of the flow cytometer used is BD LSR II( 3-laser FACS analyzer).

Before, using the machine, cells must be prepared for analysis. Single cell suspensions must be obtained. Though the process is simple, but it is very tedious and time consuming. I don’t see the significance of explaining individual steps, so if you are interested, just send me a comment.

I usually run 3 replicates for each condition(different concentration of polymer-DNA complexes added into cells). In this experiment, I have 10 condition, hence, I will have 10x3=30 samples. The whole process can take a day! I’m always super exhausted at the end of the day.

Principle of flow cytometry

Image 1: taken from http://biology.berkeley.edu/crl/flow_cytometry_basic.html

When measuring, cells will flow into a stream of fluid in a flow chamber, allowing only single cells to pass due to the reduced diameter. Lasers are often used as light source in flow cytometry. In this case, argon ion laser is used to excite the GFP cells at 488nm. The corresponding fluorescence signals are picked up by optical detectors at 503-530nm.
The signals are then ‘processed’ further, but it is so technical, so no need to explain as the machine does everything. =))

Data presentation

This is the most important part. A computer is connected to the flow cytometer to analyze the signals. It can be presented in many ways, but I only use two, histograms and dot plots.

1) Dot plots( 2 dimension)

Image 2

y-axis: SSC-A(Side SCatter): parameter that relates to the density/granulariy of the cell.
X-axis: FSC-A (Forward SCatter): parameter that relates to the size of the cell.

Both axes allow us to create a 2 dimensional plot.

Dot plots are often used to measure cell size and density. Based on both axes, it will position the cell in a form of ‘dot’. Each dot represents 1 cell. Only cells(dots) that are in the scatter gate are analyzed, while the others are ignored. The scatter gate is created by the user(me =)). Hence, it can be shifted to the right or left, or even make it larger. So how do I decide? I use controls and all my other samples are based on that.

Why do I need controls?

Reason: To properly set the conditions for flow cytometry, negative (untreated cells: no GFP) and positive controls are required for each cell type and for each fluorescence dye. Different cell types auto-fluoresce at different intensities, so a negative control for one cell type might appear positive for another cell type. In addition, if one cell type is significantly larger or more complex than the other, their forward- and side-scatter settings will differ.

In real life, dot plots are often used in hematology to distinguish different lineages of blood cells, as different type of cells will appear at different parts of the plot based on their density and size.

2) Histogram(single dimension)

Image 3

y-axis: count: no. of cells
x-axis: GFP-A: relative fluorescence intensity

This is the simplest way to present the data. Peaks that are in the right ‘box’ are GFP positive, while those in the left are non-GFP cells. Again, I must have negative and positive controls to ‘gate’ the peaks.

Finally, The flow cytometer will automatically tell me how many GFP positive cells based on the data analysed and the scatter gate I chose.

Image 4: Interpretation of data

Note: Image 2-4 are posted with permission.

Till here then.

Nisha

0503254E

TG02

Sip sharing..cytology!

Hey people! I’m now attached to a routine cytology laboratory. My tasks assigned are: mount stained slides manually, sort slides, receiving/collection of specimens (both gynae & non-gynae), processed urine and sputum samples. So this time, I will blog on the procedure for sputum processing and Papanicolaou staining.

Sputum testing is to differentiate the nucleus and cytoplasm of the cells to detect malignancy by comparing the Nucleus: Cytoplasm ratio (N:C ratio).

Procedure for sputum processing
1. Receive sputum sample
2. heck particulars of patient and nature/number of specimen
3. Assign a doctor to the patient and stamp date/time received and initialized by technician
4. Label all containers containing sample with pre-printed labels
5. Initial of technician performing sputum sample and the condition of sputum sample
6. Label the specimen lab number on the frosted end of the four clean glass slides.
7. Label on one of the slide the name of the special stain, if requested example Ziehl Neelsen
8. Pick the selected material with a Pasteur pipette and flush them onto the surface of the labeled glass slide
9. Holding a slide on one hand, pick up another slide to pull the material evenly over their surface to make smear. Avoid leaving rough or raised areas that may cause cover-slipping problems.
10. Used Pasteur pipette are being disinfected in sodium hypochlorite.
11. Place slide immediately into container containing 95% ethanol. 12.Separate out slides for special staining.
13. Additional slides are prepared if future request for special stains are made.
14. Load slides into autostainer using slide hold (rack)
15. Stain with Papnicolaou stain
16. Mount with 24 by 50mm cover slips and Depex manually
17. Place in oven for 10 to 15 minutes to let Depex dried up
18. Slides are ready to be observed under microscope

Label with specimen lab number and name of specimen
A drop of sputum sample

Making a smear using the ‘touch and pull’ method’

A smear done

0.5% Sodium Hypochlorite (prepare and use only fresh solution) is a disinfectant. It is active against bacteria, spores, fungi, viruses including HIV and HB. Concentration is 0.50%.
Used for specimen disposal, contaminated waste, materials spillage and non-metal equipments. The contact time must be at least 30 minutes for optimal effectiveness.

Routine Papanicolaou Staining
Papanicolaou method is a polychrome staining reaction (staining the cytoplasm of different cell types different colors) designed to exhibit differences in cellular morphology, maturity and metabolic activity
Intact cells in a cytologic smear tend to overlap and some appear in three dimensional configurations, the greatest value of papnicolaou staining method are the resultant transparency of the cells and clear definition of nuclear detail
Used for gynaecologic and non-gynaecologic specimens

Principles
1.Fixation
2.Nuclear Staining
3.Cytoplasmic staining
4.Clearing

A series of graded percentage of alcohol (80%-70%-50%) to hydrate the cells gradually before immersion in the aqueous haematoxylin solution
After approximately two minutes in haematoxylin the cells are then dehydrated (70%-80%-95%) prior to immersing in the alcoholic counter stains (Orange G and Eosin Azure)
Following the two cytoplasmic stains, the slides are then rinsed in alcohol

Fixation
To fix/preserve the morphological details of the cell in as perfect a condition as possible

Nuclear staining
Regressive method: The nuclei are overstained with unacidified haematoxylin; excess stain is then removed with dilute hydrochloric acid. The hydrochloric acid must be removed by a bath of running water.
Progressive method: To stain for the desired color intensity. This method eliminates decolorizing with hydrochorite acid and the need for subsequent running in a water bath. Recommend for non-gyanecological cell samples because they do not adhere to slides as well as those from the female genital tract. To cause the colour of the stain to change from red to blue, the slides may be ‘blued’ with dilute solutions of ammonium hydroxide (NH4OH, Ph 8.0-8.5), lithium carbonate ( Li2CO3, pH 8.0-8.5) or Scott’s tap water substitute (pH8.2).

Cytoplasmic staining:
After the nuclear staining, the cells are dehydrated through rinses of 95% alcohol. This dehydration prepares the cells for the two alcohol stains. First, the OG-6 stain, the slides will be in the alcohol for 1 minute. If there is any keratin in the cytoplasm of these cells, the OG-6 will stain it a brilliant orange. Following the OG-6 stain, the cells are rinsed in two 95% alcohol baths. They are then immersed in the modified EA stain for 2 minutes. The modified EA is a combination of these two stains : First is the eosin, which stains the cytoplasm of mature squamous cells, nucleoli and cilia. Second, is light green, which stains the cytoplasm of parabasal and immediate squamous and columnar cells.

Clearing:
Following the EA stain, the cells are then taken through two 95% alcohol rinses. These high concentrations of alcohol help to provide a clearer view through areas of overlapping cells
Next, the cells are taken through 10% alcohol rinses for final dehydration
Upon complete dehydration, the cells are placed in the two to four rinses of xylene where they remain until the coverslipped. The xylene will carry light rays from the microscope in the same way that the cell will, thus, making the cells transparent.

Note: Pictures are taken with the permission from my supervisor.

Yup so thats all :) do feel free to ask any questions..

Sharon Ang

0503219H

Tg02

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