A normal healthy individual should not have any blast cells in the CSF. This is used as a reference for screening of blast cells in the CSF.
Staining...
2. Then, twice the amount of phosphate buffer is added on top of the stain
3.The glass slide is left to stain for 5 mins
4. The slides are them washed and left to air dry on the rack
5. Slides are then viewed under the microscope under high power (40X, 100X)
If cells appear to be disintegrated, then the whole process have to be repeated. But if the cell population is too high, then the sample may be eluted in saline.
This whole process only takes about 15 mins! That's it for this blog entry:D
Hope u guys have enjoyed ur SIP...cya back in sch!:D
Neela
TG02
Hi, this week i will be sharing on virus amplification.
Similar to what Shu Hui has previously shared in one of her entry on how to cultivate virus by infecting cells in a flask, my lab also uses this method for viral amplifcation.
Introduction
To amplify (or multiple or to increase in the number of) a specific virus, we need to have cultures of the host cells specific to the virus. The virus is used to infect the host cells, so that it is able to replicate and increase in number using the host cell machinery. We make use of this mechanism to amplify our virus stocks, once the volume is running low, when the virus titer (pfu/ml) is too low or when the virus stock is too old for usage.
For the type of virus we are using, the virus has 3 phases in its infection cycle.
The 3 phases can be classified into the early infection, late infection and very late infection.
Also, this virus we are using has two unique morphology, one is the budded form and the other is a polyhedron form.
During each phase of the NATURAL infection cycle, certain event takes place in the host cell as follows:
Early infection (0-6 hours) : Virus enters the host cells and decodes itself
Late infection (6- 24 hours) : Replicates and buds out of host cells without lysing the cells; this give rise to the budded form of the virus
Very late infection (18-24 to 72 hours) : Single or a few budded virus comes together and gets encapsulated in a polyhedron coat; this give rise to the polyhedron form of the virus.
When the host cells eventually lyses during this phase, the polyhedron form of the virus will be released.
Note
*The virus we use in the lab is genetically modified for research purposes. The polyhedron gene is deleted from the virus, because only the budded form of the virus is useful to our research. The polyhedron form of this virus is not able to infect any cells and thus cannot be manipulated for research purposes such as protein expression or gene delivery
Therefore, even til the very late infection cycle, only budded virus will form and buds out of the host cells we infect, when this genetically modified virus is use.* However, the host cells will still lyse after >72 hours.
Method
We use tissue culture flask as shown in (picture 1)to seed the host cells. These tissue culture flask are the same as those used to culture mammalian cells which has a inner flat treated surface for easier cell attachment.
We use different sizes of the flask, depending on the scale of our amplification.
There are 3 types of flask:
T25 (Yellow) : Smallest flask (25cm square)
T75 (Green) : Medium flask (75cm square)
T175 (Red) : Largest flask (150cm square)
T25 flask requires 5 million host cells
T75 flask requires 10 million host cells
T175 flask requires 20 million host cells
Picture 1: T25, T75 and T175 Tissue culture flask, Taken from www.wwmponline.com/cart/images/T/flasks-01.jpg
Before the required number of host cells is seeded into the flask, medium has to be first added into the tissues culture flask.
This volume of medium, is just enough to cover the surface of each flask:
T25 flask requires 5ml
T75 flask requires 10ml
T175 flask requires 18ml
Then the required volume of cells (depending on the concentration no. of cells/ml) is added to the medium in the flask. The flask is held horizontally and rocked back and forth at a gentle angle of about 30 degrees. It is rock for a few times to evenly spread the cells so that the cells covers the whole surface of the flask as it begins to attach to the treated surface of the flask. These flask are incubated for 1 hour (at 27 degrees celcius, 5% carbon dioxide incubator) for the cells to attach.
The cells are incubated at 27 degrees celcius because it is a non-mammalian cell.
The flask cap has a unique 0.22micrometers membrane, which allows carbon dioxide to diffuse into flask.
After the cells attach it looks like this:
This is just one small part of a T175 flask under 10x magnification
Picture 2: Virus Host cell (Taken with permission)
After the cells has attached to the flask treated surface, a certain volume of the medium is pipetted out to concentrate the cells in a smaller volume. This would allow the virus to attach to the cells more easily later, when the virus is added.
T25 leave behind 1ml
T75 leave behind 3ml
T175 leave behind 5ml
Add ~100microliters of the virus stock (the current one that we have) to the concentrated cells. The volume to add can vary, depending on how old the virus stock is. A older virus stock may have a lower virus titer (pfu/ml), so we can add more volume such as ~200microliters .
These flask are then put on a rotator (for 2 hours) which continuously rock back and forth to allow the virus to find and attach to the cells.
After the virus attach to the cells (assuming, because we can't see any changes yet), we add more medium to each flask to maintain the infected cells in the flask.
T25 add 5ml
T75 add 10ml
T175 add 18ml
The infected cells are incubated for 72 hours (at 27 degrees celcius, 5% carbon dioxide incubator), so that the cells are now in the very late infection cycle.
These are how the cells look like during the very late infection cycle: under 10x magnification with 1.6x aperture
Signs to take note of: Cells look swollen, has black dots in the centre of the cells, uneven surfaces of the cells and some cells have even fuse together.
Picture 3: Infected virus host cells after 72 hours (Taken with permission)
When these signs are observed in the very late infection cycle, we assume that majority of the viruses has bud out of the infected host cells, (virus are too small to be seen under our phase contrast microscope. It can only be seen unless a high power electron microscope is used) and it is very likely that the cells are going to lyse soon. Virus has to be collected before the cells lysed. When the cells lysed, enzymes released can cause break down of proteins found on the surface of the virus and lowers the virus titer greatly.
Virus collection
Since the cells are attached to the flask surface, as the viruses buds out of the infected cells, it is released into the medium. Therefore, virus can be collected by pouring the medium from the flask into a 50ml tube. The infected cells remains attached to the flask surface and are discarded.
This tube is spin down at 1000rcf for 10minutes to remove any cells debris (from a small proportion of infected host cells that may have detached). The supernatant is collected in a new 50ml tube without disturbing the pellet of cells debris. The new tube is wrapped with non-shiny side of the aluminium foil (because viruses are light sensitive), and stored at 4 degree celcius for future use.
TG02
Okie, time to blog again. I am attached to the biochemistry lab for this two weeks. During the campus discussion week, i've shared about G6PD deficiency testing. Now, i am going to blog on another test done in the biochem lab.
Except G6PD, most of the things done here make use of machines for testing. Neonatal Total Bilirubin is one of them that uses machine to analyse the results.
Background infor
We've learned how bilirubin is formed in school already. In case some of us forget, here is brief explanation of the process. Haemoglobin in the body is broken down into heme (iron and porphyrin) and globin (protein). Iron and protein are then re-utilized by the body while heme goes through a metabolic degradation where the porphyrin ring opened up to form biliverdin. Biliverdin is then further reduced to bilirubin, where it is transported to the liver via the plasma. It is in the liver where it conjugates with glucuronic acid to form bilirubin diglucuronide and excreted into the bile and then goes into the intestine. It will then reduced to form urobiliogen by the action of intestinal bacteria.
So, in this test, the sample that is collected is blood. The blood has to be spin down such that the plasma could be obtained to test for the bilirubin level.
Clinical Significance
When blood contains excessive bilirubin, it gets deposited in the tissues and cause the shade of yellow. It is also referred as jaundice or icterus. This condition may be due to 3 reason.
1) The bilirubin produced is more than the kidney can process and excrete. ( Hemolytic disease)
2) Having liver damage which resulted in inability to excrete normal amount of bilirubin produced.
3) The liver’s excretory ducts are obstructed and blocked.
Jaundice in the infants are quite common as their liver are immature or inefficient. Due to the deficient liver enzymes, the liver could not conjugate the bilirubin. The condition gets better when the liver is more matured and the enzymes are more functional. Possible treatment for the babies are phototherapy.
The bilirubinometer is used to measure the total bilirubin in the plasma of newborns. By measuring the absorbance of the plasma at 460nm, the concentration of bilirubin can be determined.
Methods
1) A heel prick is done and blood is collected via the capillary tube through capillary action.
2) A capillary tube is used to collect the blood of the newborn. When it is received in the lab, make sure that one end of the tube is properly sealed.
3) The capillary is placed in a plastic tube in the centrifuge in spin down at 3000rpm for 3 minutes. (make sure that one end of the tube is sealed and sealed end placed facing down)
4) Plasma should be visible at the top of the tube.
5) Spring-lock the capillary into a dispenser and slowly inject the plasma into a clean cuvette. ( Make sure there is no air-bubbles)
6) The capillary tube is then discarded and the cuvette is loaded into the bilirubinometer.
7) The measuring chamber will rotate downward to align the plasma of the patient to the optical path and results will be shown.
8) The results are to be recorded on the request form for entering into the system later on.
* If the reading exceeds the critical value of more than 300µMol/L, the test have to be repeated.
That's all folks.
Zhenling
TG02