My 1st week of SIP in 2 Departments!!! Double the FUN!! =)

(This posting is for this week)

By Rachel,


Wow! My first week of attachment was filled with so much fun and laughter. I was posted to 2 departments.


The first 3 days was at Histology lab. I get to see different organs everyday in the trimming room and was lucky to observe a post-mortem. I would also like to clarify certain things; there is nothing scary when looking at these real organs. Though they may not be pretty to look at but they will just ‘arouse’ your excitement. Anyway, we have so much to learn from this department. =)

For a start,

Contents of my post:
1) Briefly describing the daily routine work in Histology Department
2) Operation of the Tissue Processor
3) Principles of the common stains


Daily work routine

1) Organs and other specimens are received
2) Pathologist dissects the organs and records the abnormalities observed (includes the measurement, lumps or enlargement of cells)
3) After dissecting, the smaller pieces of tissue were placed inside a labelled block for fixation.
4) Labelled blocks were placed in the tissue processor also known as Shandon Excelcior using a rack.
5) The tissues were taken out from the processor.
6) A medical technologist would embed the tissue blocks and cool them in an ice bath. (Reason: Ice bath is important to cool the wax tissue block otherwise the sectioned tissue would not be of equal thickness.)
7) Tissues were sectioned using a microtome and placed in water bath to check for presence of tissues. Slides were used to fish the tissue.
8) Slides were stained.

Note: The detailed procedure of fixation, embedding, sectioning, fishing and staining will not be described as they are similar to what we were taught before.


Tissue Processor


















Fig 1: Tissue processor
Taken on 23 June 2009

Retrived from http://www.fishersci.com/wps/images/domain/Healthcare/shandon_excelsior_ng.jpg












Fig2. A diagram of 9 bottles from the computer screen of the tissue processor


Description of Fig 2:
· A1 – A6: These containers contained alcohol.
· X1 - X3: These containers contain xylene
· A1 alcohol is considered as the ‘dirtiest’ alcohol and A6 is the ‘cleanest’ alcohol.
· X1 alcohol is considered as the ‘dirtiest’ xylene and X3 is the ‘cleanest’ xylene.






















Fig.3 Diagram indicating the temperature and pressure within the tissue processor as well as the different containers filed with the various reagents


Description of Fig 3:
· Alcohol quality: When the arrow reaches or is within the red block, this meant that the alcohol used is in poor quality.
· Pressure within the green block meant that there is still air present inside the cubicle.
· Temperature is an indication of the humidity of the cubicle.
· WW represent waste wax. This is to store the poor quality wax used and be manually removed.
· W1-W3 contains wax. W3 is the ‘dirtiest’ and W1 is the ‘cleanest’ wax.
· FIX 1 and 2 contains formalin.
· Ex 1and 2 are used for refilling the alcohol or discard the poor quality alcohol.
· F1-F3 are used for washing the materials.
· F1 contains xylene, F2 contains alcohol and F3 contains water.

Process involved in the tissue processor:
1) Formalin was added and filled the cubicle.
2) After an hour, formalin removed
3) A1 was added and filled the cubicle. If A1 alcohol decline, it will be discarded into the waste bin automatically. Afterwhich A2 will replace A1 and so on. Manually added alcohol will replaced the empty container A6.
4) Followed by X1 was added and filled the cubicle. If X1xylene decline, it will be discarded into the waste bin. Afterwhich X2 will replace X1 and so on. Manually added xylene will replaced the empty container X3.
5) Hot wax was added and filled the cubicle. If the wax quality decline, it will be replaced by new manually added wax.
6) The whole process takes about 14 hours. The tissues will then be considered as appropriately fixed.


Principles of the Different Stains

The common stains are Haematoxylin and eosin stain (H&E), Periodic acid–Schiff (PAS), Periodic acid–Schiff diastase (PASD), Prussian blue (PB), Ziehl-Neelsen stain (ZN) and Mucicarmine stain.

PAS and PASD are used to observe glycogen in the cells. PB and mucicarmine stain is to observe iron and mucin respectively. Other stain such as immunohistochemistry (IHC) is used only when necessary.

Principle of PAS stain is use in the demonstration of basement membrane such as glycogen, fungi, mucin, secreting adenocarcinoma and mucosubstances secreted from the epithelial of various organs. Periodic acid oxidises the carbon-carbon bond. This forms aldehydes which can react with fuchsin- sulphurous acid to form magenta colour.

Principle of PASD stain is to demonstrate glycogen. This is carried out by 2 slides. One was treated with diastase (an enzyme). This enzyme will break down glycogen to form maltose and glucose which will eventually be washed out with water after treatment. Areas of PAS positive shown on the untreated section that appears PAS negative on the treated section. This meant that there is presence of glycogen. This is for the demonstration of neutral mucin which is not digested by diastase and will stain with PAS.

Principle of Prussian blue It is used to stain iron which is normally found in spleen and bone marrow. Excessive amount present in hemochromatosis with deposits found in liver, spleen and lymph node. A reaction occurs when section is treated in acid solution of ferrocyanide. Iron reacts with ferrocyanide and produces a bright blue pigment (ferric ferrocyanide).

Principle of ZN stain
It is to stain acid fast bacteria such as tuberculosis. Lipid capsule of the acid fast organisms takes up Carbol-Fuchsin stains and resists decolourization with a dilute acid rinse. Lipid capsule of the mycobacterium of such high molecular weight is waxy, at room temperature and successful penetration by aqueous-based staining (such as Gram’s) is prevented.

Principle of IHC is to visualize Ag via binding of primary Ab to Ag, followed by addition of secondary Ab which binds to primary Ab. An enzyme complex with a DAB substrate produces a brown colouration upon reaction with the Ab.

This is about all for now. If you have any queries, please feel free to ask me.=)

My next post is CYTOLOGY!!! Another interesting subject regarding cervical cells ……

1st week of SIP,WOOO!

DONE BY: JOANNA YEO, WOO!

First week of SIP was definitely an exciting experience!

For the first 3 days of SIP, I was attached to the Cytogenetic department whereby i was taught the roles and importance of Cytogenetic, which is mainly to detect abnormal chromosomes to provide the right diagnosis.

The specimen type processed are prenatal(amniotic fluid, chorionic villi, fetal cord blood), neonates or adults peripheral blood, product of conception(miscarriages), haematological malignant diseases ( bone marrow, tumour tissue).
So before the chromosomes are actually identified and analysed, we actually have to culture and harvest the cells first.

The standard workflow is specimen procurement->culturing of cells-> harvesting (mitotic arrest, hypotonic treatment, fixation -> slide making-> chromosome staining-> Karyotyping

The culturing techniques are similiar to that of MCT techniques (such as changing of medium, incubation, tiffing, subculturing, etc) except that in the lab, we seldom use flask. Instead We use in-situ cultures whereby we grow the cells on cover slips and placed them in very small petri dishes(They are cute! haha! 1/4 of the size of normal petri dishes) Reasons being for easier identification.

Also, remember during the changing of medium we have to use our pipette manually to suck out the media? In the lab they use vacuum to suck out the media which I think it’s really convenient as there are lots of samples and this will save up a lot of time as it is fast and simple! After culturing, we have to observed the colonies under the microscope to see if there are enough colonies for harvesting and to ensure optimal mitotic index. For example, amniotic fluid should have at least 5 colonies of smaller size if not,it might get too dense and causes mitotic index and spreading to decrease.

So, the brief steps for culturing techniques I’ve learnt are:

Day 1: Specimen comes in as a syringe(20ml), it is then equally divided into 2 tubes(one serving as a back-up), the other tube is then further divided into 4 coverslips. (2 coverslips for each incubators.) Cover slip can hold 0.5ml of cell suspension.
Day 2: Do Nothing
Day 3: Add 1.5ml of media( Flood coverslip) (use of different media for coverslips in different incubators)
Day 4: Do nothing
Day 5: Suck out media and add 2ml of media
Day 6, 7: Hold; Check for harvest
Day 8: Feed

After which , keep checking for harvest, if not change media every 3 days.

Things to note:
-Do not dish violently to prevent detachment of cells.
- Usually feed before check for easier identification under microscope.
-Use same pipette for same cases

After which, is selection for harvest. Monolayer cultures with 70-80% confluency usually yield ideal mitotic index. To stimulate mitotic index, cultures should be fed 24-48 hours before harvest to increase mitotic index(MI).

For amniotic fluid, in-situ harvest shold have at least 5 colonies of small size. Large colonies should be avoid as to prevent high density and reduction of MI and spreading.

When cells are ready to be harvest, reagents( colcemid) which is a mitotic inhibitor is added to arrest cells at metaphase stage. It acts by depolymerizing microtubules that make up the spindle apparatus thus, preventing cell division.

Brief steps for Harvesting of cells consist of

1) Hypotonic treatment
-Causes water to go inside the cells, which in turn causes chromosomes to spread out, so that we they don’t overlapped and we can identify them easier)
- Common solution used: 0.075M KCL or 0.8% Sodium citrate or mixture of both.

2) Fixation
- Use of 3:1 methanol: glacial acetic acid
- Kill cells, fix membrane
- Precipitate proteins, and remove H1 fraction of histone proteins from chromosomes
- Precipitates free Hb from RBC lysis after hypotonic treatment
- Fixed membrane are brittle, causing cell membrane to rupture and disperse during slide dropping procedure

3) Drying of slides in drying chamber

After drying, slides are heated for one and a half hours prior to staining.


The interesting part comes!!! After harvesting, it is to identify chromosomes and pair them up! (Karyotyping) Oh yah if you guys are interested, I can let you guys see my exercises! =) So basically, we capture a pool of raw chromosomes(under the microscope which is connected to computer and camera) and try to pair them up. Then compare with the standard and normal chromosomes to see if the person have any genetic abnormality.




Retrieved from http://en.wikivisual.com/images/5/53/NHGRI_human_male_karyotype.png.

Above is an example of normal male karyotype.


Examples of genetic abnormalities are:
- Males with extra X chromosome resulting in Klinefelter syndrome (incidence is 1 in 1000)
-Females with Monosomy X resulting in turner syndrome( incidence is 1 in 5000)
-Trisomy 13 resulting in Patau syndrome
-4p- resulting in Wolf-Hirschhorn Syndrome

Sorry for the long post! I try to cut short already! hahhaas. if you guys are interested in steps for staining and slide making you can ask from me! ((((((((((((:

do feel free to ask if you have any queries


CHEEERIOSSSSSSSSSSSSS! *****

Micro Week 1!!! WOOHOO~!

Done By: Janice Yeh

Dept: Microbiology
Section: Urine Feme

Hello girls! and people reading the blog for the first time!
just did some tidying up of the blog. :D
like the picture on the right girls!? haha.

I'm currently in the Microbiology Department of my company.

it has been a very fun and enriching time honestly and it helps when you have very nice people working around you! :D

arriving at the company on the first day was a little tricky cause we didn't know how. haha.

when we reached there, we had a walk through the entire company to the various departments.

then i was sent to the microbiology lab, got introduced and before i know, i was already helping them out with the samples that came in in huge bulks.

There are mainly 4 sections in the lab:

Urine Feme, Culture, Stool Sampling and Culture and Bacteria Identification

this week, i am under Urine Feme, the basics of microbiology work

which is to deal with urine samples that come in everyday... i mean monday to sunday!

AND YES... i have to work on saturday as well!

so here's mainly what i did this week...

Urine Feme:

- mainly testing for Urinary Tract Infection, Renal or Liver Disease, metbolic disorders

- by 2 methods, Chemistry and Microscopic

- Chemistry involves using a dipstix (Combur Test M) and Urisys 2400 Analyzer (below)
- Microscopy s to clarify the results of physical and chemical findings by looking out for traces of

WBC, RBC and Epitherlial cells,etc...after which, quantifying them.

Urinsys 2400 Analyzer
Retrieved on 27 June 2009, from website: http://www.mylabonline.com/products/urinalysis/2400.php

Procedures:

(A) At the Start of the day- Starting up of Analyzer

1. Input Zero to delete and remove all the history results of the previous day
2. Ensure that the screen shows '0000'
3. Chek quantity of Biochemistry Urine Sticks Left, if there are no more sticks left, which will be indicated by the alarm of the analyzer, the cassette has to be changed (below)

Urisys 2400 Casette
Retrieved on 27 June 2009, from website:
http://www.mylabonline.com/products/urinalysis/2400.php

(B) Preparation and Running of Controls

1. Aliquot 31 tubes of about 2.0-2.5 ml of Liquichek Urinalysis control level of 1 and 2 control reagents respectively into the 5ml plastic tubes and store between 2 to 25 degrees celsius
2. Stability of reagent is 30 days. so after 30 days, need to prepare samples again.
3. Allow the reagent to stand for 1 to 2 mins after removing it from the fridge.
4. Transfer the reagent into the conical tubes and load them onto the rack
5. Place the rack on the analyzer and press start

(C) Sample Preparation for teststrip analysis and Microscopy:

1. Label the sequence number on the 10ml conical presure tubes.
2. Transfer the extra barcode on the urine container to the conical plastic tube and label the sequence no. on the cap.
3. Gently invert the urine sample several times to mix well.
4. Pour abt 3 ml of urine into the conical tube.
5. Load the ubes into the sample rack
6. Check the sequence no. on the menu screen to ensure it tally with the tubes on the rack
7. Press start on the menu screen to run the samples
8. Results will be printed out by an assigned printer alongside the analyzer
9. Remove the tubes from the racks and place 20ul of urine sample into a chamber on the KOVA slides and observe under light field microscope
10. Count and quantitate the cells and other elements found
11. Tally with the results

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I was also given a test (informal) to see if i am able to identify the various elements under microscope. thankfully, i didn't do too bad.

Microscopic Observations:
Here is my little trick of recognizing them under the microscope:
Crystals:
1. Calcium Oxalate Crystals - they look like 'little envelops' or 'circular', they can also be found to overlap each other
2. Uric Acid Crystals - they look like 'lemons' or in a shape of an 'eye', are uaually yellowish under low power observation

Blood Cells:
1. Red Blood Cells - other than it does not have a nucleus, it appears often in chains or overlap, or some may clump together (2-3).
2. White Blood Cells - bigger than RBC, has a nucleus, not in chains.

Microorganisms:
1. Bacteria - most commonly present in rod shape, under low power observation, looks like a threads.
2. Yeast - circular; joined together
3. Parasites - looks like WBC but larger

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Overall, there was alot of hands on experience and it is surely a very busy week.
i'm still getting to use to working on saturdays and shifts from 1-9pm. hmmm...
very tiring.! hahaha. but worth the experience. :D

the fun part of the lab?? we took photos on fridays....randomly!!! hahaha!.... don't get the wrong idea, it's my collegues camera, not mine! ha! we had a 'dress-nicely' day. which was saturday! haha...
fun fun fun! but i still miss sch! really....somehow.....it's different lah!

looking forward to tmr! a new week, a new horizon of experiences. :P

Haematology Routine Lab!

Department: Haematology
Section: Routine Lab

Hello! I'm Vanessa! Today i will tell you all about the workflow of the haematology routine lab, the one i work in this few weeks!

First, there will be different kinds of tubes of blood samples delivered to the haematology department reception. Then there will be someone to seperate the tubes for different purposes! There are different section in the haematology department, they are, routine lab, coagulation, Hb electrofluoresces and many more.

For the routine lab, tests done are:
-Complete blood count (CBC)
-Differential count
-Reticulocytes count (retic)
These are all done by machines, sinces its a lot faster The brand of machine use in my lab is Advia. I was told that, if a full blood count is ordered, i will press CBC/Diff option, if only Hb count is ordered, i will press the CBC option, and if retic is order, with the full blood count, CBC/Diff/Retic is selected, without full blood count is just the Retic option.

Then the machine will do its stuffs, and results will be transmitted to the computer, which will be verified by us. Verifying means checking if MCHC is above 37 and platelet count is below 140. If one of the value is out, it can mean that there is clotting of the blood, which could be caused of uneven mixing of the EDTA with the blood. So we will have to check for clotting using 2 wooden sticks to stir the blood to check if there is clot particles. And also, to do delta checks, which means comparing the results from previous test done and there is more than 10% difference between today's and last time's result, so we will have to highlight this by writting a comment in the results to inform this big change to the doctor so that things would be done.

Other test that is done are:
-Peripheral blood film
-Thalassaemia
-Malaria parasites
-Retic blood film
-ESR (erythrocytes sedimentation rate) and many more.

For the normal full blood count ordered, a blood film will be done, by putting a drop of blood on a slide then using a slider, hold in a 45 degree angle, smear the blood uniformly, creating a fading tail (most favor viewing part) and read using micrscope.

For thalassaemia, a drop of blood will be mixed with a drop of Brilliant Cresyl Blue, and is incubated for an hour before it is smeared on a slide then viewed.

For testing of malaria parasites,besides the routine test done. 2 extra thick flim is needed. This is done by using 2 stick and spread the blood onto the slide in a circular motion. The slide is then air dried or dried by placing on hotplate then stained using diluted Giemsa stain, and lastly viewed.

For retic blood film, a drop of blood is mixed with brilliant cresyl blue stain and is incubated for 15min before it is smeared and viewed.

For ESR, blood will be poured into a sodium citrate (anticoagulant) dilution vial. There is a line so that the amount of blood poured will be mixed with the sodium citrate with the correct dilution times. Then the ESR pipette is pushed into the vial via a breakable opening and is left on a rack for an hour horizontally, then read.

All the results and findings will be combined and sent to the doctor, where the preliminary diagnosis will be confirmed.

Thats all for now!

The very 1st week

Composed by: Vo Thu Hong Anh [Jess Vo]

Wohooooooooooo........I'm back to this company for the 2nd attachment, this time, things are getting really serious because it's now my real internship which will last for 5 months ^^

I have been assigned under Cell and Tissue Engineering Department. After listening to my mentor's explanation about the project, I am so amazed by the scientists' ideas in the approaches of diagnosis and treatment of liver diseases via the study of microRNA.

Well, I have been quite busy for the first week as my mentor and his colleague showed me the proper techniques to culture cells, to perform Western blot and to carry out RT-PCR. After that, I was allowed to subculture cells on my own under their supervision as well as to assist them in performing Western blot and RT-PCR, such as helping them prepare the reagents needed for the experiments.

The techniques for the cell culturing are almost similar to what we have learnt in MCT subject. Moreover, there are 2 important things which I have noted:
1) Must wear a different lab coat when entering the cell culture lab
2) The only thing which is 100% sterile in the laminar flow hood is the air, that is why we need to place the caps of the bottles, flasks, tubes facing up =D
And you know, the laminar flow hood we used are Class II, which provides protection to the cultures as well as the technologists, yeah yeah!

Oh yah, you guys remember the time we did Western blot during MBio practial? We had troubles with finding the wells on the gel plate and were scared of adding the reagents out of the wells yeah? In my lab, the gel plates used are commercially prepared and there are markers on the plate indicating where the loading wells are, so it's really easier to see the wells while loading in the samples, ahhahaha.

For RT-PCR, not much too talk about, except for the preparation of the reagents.........it's really tedious, oopz!

RT-PCR machine ABI Fast 7500
http://www.azcobiotech.com/images/7500.png

96 well-microplate for RT-PCR
(it's slightly different from the plate that we used for ELISA in our school ^^)

https://products.appliedbiosystems.com/ab/en/US/htdocs/productMgr/images/Product-shots-003_thumb.jpg

Over all, my project is related to most of the subjects in year 2, so I'm kind of feeling worried of forgetting year 3 subjects' knowledge and lab techniques after 5 months of SIP, lolz.

So yah, I have learnt quite a number of lab techniques during this week, I hope that I will be allowed to do the whole experiment on my own soon, hahahaha. Anyway, there is a lot of principles and theories behind each experiment, so I think I gotta read up more in the next few weeks #_# lolz. Ayoh, I somehow miss TP a bit, lolz, don't you all feel the same? hahaha. Okie then, hope that all of you are doing well at your attachment places. JIA YOUUUUUUUU! [=^.^=]