Week 14 - Virology

This is the 14th week already. For the past 4 week, I was at the Immunofluorscence section. This area is mainly on antigen and antibody detection. For antigen detection, it is to detect the particular antigen that is present on the cell. There are 2 methods: direct and indirect.
For direct method, a fluorescent-labeled primary antibody is added and it will react with the antigen. Thus, fluorescence is observed.
As for indirect method, it will employ the use of a specific primary antibody (unlabelled) and a labeled secondary antibody (labeled with fluorescence dye). Therefore, antigen present will bind to primary antibody. And when labeled secondary antibody (also known as conjugate) is added, it will then bind to primary antibody and thereby staining the positive cells. The positive cells will then fluorescence under the UV microscope.
This method is more specific, thus reduce non-specific binding.

As for the antibody detection, it is usually to detect the presence of antibodies in serum.
Commercial antigen slides will be used, which means that the slides are already fixed with the specific antigen. If the corresponding antibody is present, then it will bind to the antigen. Following that, conjugate labeled to secondary antibody is added and it will bind to the primary antibody. Fluorescence will be observed when viewed under the microscope.

Now, I will know talk about the detection of Cytomegalovirus antigen by using cytospin. This is the most common test that is requested for in IF section. The purpose of the test is to identify the lower matrix protein pp65 of cytomegalovirus in the white blood cells. The principle of this test is mentioned above (antigen detection by indirect method).

Cytospin processing for CMV antigen

Processing the blood

1. Transfer the blood (in EDTA tubes) into sterile plastic centrifuge tubes
2. Add separation solution or 6% dextran, ¼ of the total volume of blood to the blood.
3. Place the tube in 37 ̊ C waterbath to allow the separation to take place.
4. Transfer the upper layer of suspension ( contains serum, plasma and leucocytes) into another sterile plastic centrifuge tubes.
5. Centrifuge the tube at 300g for 10 minutes.
6. Discard the serum and add cold water to lyse the red blood cells.
7. Vortex to mix well before adding phophate buffered saline (PBS).
8. Centrifuge at 300g for 10 minutes again.
9. Discard the supernatant, leaving the cell pellet behind.
10. Add an appropriate amount of PBS to the cell pellet and mix well.
11. Determine the white blood cell concentration using the coulter counter.
    
    Generating the cytospin slides
12. Vortex the tube before inoculating 0.2ml into the cytospin funnel.
13. Balance the funnel in the cytocentrifuge container and spin at 800rpm for 5 minutes.
14. Remove slides and dry it on slide warmer before fixing.
    
    Fixing the slides
15. Fix the slides with in fixative solution for 10 minutes.
16. Rinse the slides 2 times with PBSA.
17. Immerse the slides in permeabilization solution for 5 minutes.
18. Rinse the slides 3 times with PBSA again.
19. Dry the slides on slide warmer.
    
    Staining the slides
20. Add monoclonal antibody using a dropper, ensure that it is well spread (Vortex mab before use).
21. Place the slides in a moist chamber and incubate at 37 ̊C incubator for 35 minutes.
22. Immerse the slides in PBS to wash for 10 minutes, before placing it on a shaker.
23. Dry the slides on the slide warmer.
24. Add conjugate to the slides using a dropper (Vortex before use).
25. Repeat step 21-23.
26. Add mounting fluid and mount it with a coverslip.
27. View the slide under fluorescence microscope and record the results

Separation solution or 6% dextran - Acts to separate out the plasma and red blood cells.
Fixative solution- Fix the cells on the slides
Permeabilization solution - Permeabilise to allow antibody to access to intracellular structures of the cells and maintain the morphological characteristics.
Moist chamber- to maintain the humidity condition for incubation


Possible problems encountered:

• The cytospin funnel and chamber were not assembled well. Hence, the cells were not properly fixed as a circle on the slide. This may interfere with the interpretation of results.
• White blood cell concentration is too low, due to high dilution with PBSA. The resolution is to centrifuge the tube again to obtain cell pellet. Then, thus time a smaller volume is added to dilute the cell pellet.
• Cross contamination with other sample. Hence, to minimize this, always work with 1 cell line. And keep the other chambers away when loading cell suspension for 1 sample (As 1 or few drops may splash into other chamber).

For more information on cytomegalovirus, please refer to this website;
http://www.dhpe.org/infect/cytomegalo.html (same as my previous 1st entry)
http://www.cdc.gov/cmv/

That’s what I have for now. Thanks for reading!!

Shihui
0607135A

Week 13 – Medical Microbiology

Topic: Histoplasma capsulatum

This week I will be sharing about Histoplasma capsulatum. Histoplasma capsulatum is a yeast type of fungus that primarily infects the lungs and may spread to other parts of the body. It causes an infectious disease known as Histoplasmosis. Some of the signs and symptoms are fever, headache, chest pain, dry cough and chills.

Immunodiffusion method is used to detect histoplasma capsulatum antibodies in patients’ serum. Patients’ serum and the control antigen are placed in opposite wells and diffuse outward into the medium. To a point of equivalence concentration of the antigen and antibody, two visible precipitin lines (M and H) are formed between the two wells. M line forms near the antigen well while H line forms near the serum well.

Materials and Reagents used:
1. Glycine buffered agarose agar: a diffusion medium that does not support the growth of molds.
2. Positive control: antiserum that contains H and M antibodies specific to histoplasma capsulatum.
3. Negative control: antiserum that does not contain any specific antibodies.
4. Control antigen: Contains H and M antigens from histoplasma capsulatum.

Specimen: Serum

Methods
1. Spin down the specimen at 3100rpm for 15mins.
2. Place the template underneath the Petri dish.


3. Using a hollow pin to punch the agar and a pointed stick to pick out the unwanted agar to obtain 7 wells.

4. Add about 8ml of each reagents into the respectively wells in the following order:

a. Firstly, add positive control into wells #1 and #4.
b. Negative control into well #6.
c. Patients’ serum into wells #2, #3 and #5. (Depending on the number of specimens requested for this test)
d. Lastly, add control antigen into the centre well.

[Add the reagents in this order so as to prevent any contamination that may lead to false results.]
[Prevent overflowing from one well to another as this may also lead to false results.]

5. Place the Petri dish in a moist condition at room temperature for 23-25 hours.
6. Examine the agar over a light source. (Eg. Under a microscope)


Results


Positive controls: Two lines formed between wells #1 & 4 and the centre well. [This test is valid]
Negative control: No lines formed between well #6. [This test is valid]
Patient 1 (well #2): Positive reaction as two lines are formed between well #2 and the centre well.
Patient 2 (well #3): Negative reaction as there are no lines formed between well #3 and the centre well.
Patient 3 (well #5): Positive reaction as M line is formed between well #5 and the centre well.


LeeJin
TG02

Answers (Bone Marrow Lab)

To Lyn,

Not all 3 smears have to be done. It depends on what the doctor orders. There are 2 parts to the bone marrow aspiration. The first is to get the aspirate and the second is to get the trephine sample. If the doctor orders for just the aspirate, the med tech only has to do the aspirate and the squash smears. If the doctor orders for both the aspirate and the trephine sample then the med tech will have to do all the 3 smears. Below are the pictures of the 3 different types of smears. (taken with permission from my supervisor)

Picture 1:
aspirate smear (with fragments)


Picture 2:
imprint smear (of trephine sample)


Picture 3:
squashed smear (from aspirate with fragements)


To Leslie,

Trephine is just the name of the instrument used to obtain the bone marrow biopsy. Items include:
- a 10 cc/mL syringe to inject the local anaesthetic.
- a 20 cc/mL syringe is used to withdraw the aspirate.
- 2 needles, 1 blue and 1 green. Blue is to withdraw the local from the bottle. Green is to inject the local into the biopsy site (hip bone area)
- Iodine and septanol to clean the biopsy site
- 2 tubes of local anaesthetic (1% xylocaine), in most cases. Some patients with low threshold of pain, in other words, unable to tolerate pain as well as others, will need more tubes.
- 1 dressing set
- 1 aspiration set which contains a special large-bore needle which is inserted into the hip bone area, the extraction cannula (trephine), a marked probe is used to check the length of the sample and also to expel the trephine sample from the trephine (extraction cannula).


To Yumei,

Few particles as in the fragments present in the aspirate. As shown is picture 1 above, those round things are known as fragments.
ADDITIONAL INFO: When the doctor withdraw the aspirate, there must be fragments present, or else it is rejected and the doctor will have to re-withdraw the aspirate again. If there really are no fragments present, just the blood or the doctor is unable to withdraw any aspirate from the patient, the doctor has no choice but to get another trephine sample and put it in the tube containing lithium heparin (for cytogenetics) and add 0.9% NaCl to act as a medium.


To Ben,

Myelodysplasia is a disorder of bone marrow stem cells due to ineffective hematopoiesis. Patients suffering from myelodysplasia may progress to acute myeloid leukaemia (AML) within months to a few years. It can be detected by doing a blood test. In most cases, there is a great decrease in RBCs, causing fatigue and pallor (anaemia). Rarely, there is a decrease in platelets which will lead to bruising and bleeding or a decrease in WBCs which will lead to fever and infection. At times, nonspecific symptoms like loss in weight and appetite may occur. It is because of the abnormal CBC results that lead to the decision of performing a bone marrow aspiration on the patient.


To cornelyus,

Yup, maygrundwald-giemsa stain contains methanol. Methanol is used to fix the smear to the slide so when it is washed, it will not be washed off.


To quan jun,

1) yup, I got to observe the bone marrow aspiration LIVE. =)
There are 2 parts to the procedure. The first part is to get the aspirate. The second part is to get the trephine sample. First, iodine followed by septanol is used to clean the biopsy site. Local anaesthetic is administered to numb the site. Then, a special large-bore needle is inserted and rotated. A syringe is then attached to the needle and the aspirate is withdrawn. For the second part, the needle is inserted further inside. A marked probe is inserted to check the length of the sample and removed. An extraction cannula is inserted into the needle cannula. The needle is then rotated and the extraction cannula is removed from the needle. The marked probe is used to expel the trephine sample from the extraction cannula.
As for the reagents used, I’m not sure what u meant. Or do u mean items used to carry out the aspiration? If yes, do refer to my answer to leslie above.

2) as for bone marrow fragments, do refer to the picture 1 above. They are fragments found in the aspirate from the bone marrow.

3) for this job-related question, I’m not sure which section u are referring to. But since u mentioned about processing samples, I’ll presume it’s the routine and coagulation section. For the routine section, we can process up till 250 to 400 samples a day for normal working hours (8am to 4pm). Monday is always the busiest day. After 4pm, the samples are left for those who are doing payback or night shift. There isn’t really a time limit given to process each sample. As long as there are samples received, we will just process it. During working hours, if the result of the sample is required by the doctor urgently, the samples are sent to the STAT lab.
For the coagulation samples, we can process up till 100 to approximately 200 samples a day during working hours (8am to 4pm). Urgent and non-urgent samples are run together. However, if the sample is really urgent like from the A&E section or so, we will try our best to run it first.


MALERIE (not malarie) =)
TG02

Week 12 - Bone Marrow (Haematology)

I was attached to the Bone Marrow Lab for 3 weeks. Basically, the job of a medical/lab technologist is to get the aspirate and trephine sample (bone marrow biopsy) which the doctor extracted from the patient’s bone marrow and prepare the smears on the spot. Blood taken from the patient’s finger, pricked using a lancet is also smeared on the spot. The technologist is also responsible for letting the doctor know how much aspirate or trephine sample is needed and place it in the respective tubes and bottles. How much aspirate needed depends on the tests ordered by the doctor on the request forms.

After that, the slides are brought up to the lab where they are stained, dried, labeled, mounted and examined under the microscope. Patients’ information such as the name, hospital, ward, bed no., diagnosis, gender, age, etc. are recorded into the record book and also entered into the LIS.

During my 3 weeks there, I was able to observe bone marrow aspirations and also helped the doctor prepare the items needed for the aspiration when the nurse is busy with something else. I also helped my colleague place the aspirate and trephine sample into the respective tubes and bottles and ensured that all the required forms and tests requested by the doctor are ticked. After about 2 to 3 patients’ procedures are completed, I brought the slides up to the lab where I stained, labeled and mounted the slides and also recorded the required information into the record book and forms. During my free time, I practiced doing the various types of bone marrow smears except for the imprint smear as we do not have any extra trephine sample.

Preparation of smears

The 3 types of bone marrow smears to be prepared are:

1) Aspirate smears:
- Aspirate must contain bone marrow fragments.
- Same way as preparing a PBF smear.
- Accept only when marrow fragments are found mostly at the tail of the smear and free marrow cells can be seen in stained films.

2) Squashed smears:
- Few particles are placed on the centre of a clean slide.
- Another slide is used to gently compress to spread and disperse the particles as the slides are pulled apart.

3) Imprint smears:
- Bone marrow trephine biopsy sample is gently touched and rolled along between two clean slides.

The purpose of doing a bone marrow examination by preparing these 3 types of smears is to be able to show the different cells present in the patient’s bone marrow in order to diagnose certain conditions, assess the progress or stage of the patient’s disease and also to monitor treatment of certain conditions. Conditions include multiple myeloma, leukemia, lymphoma, anemia, amyloidosis, polycythemia vera, myelofibrosis, etc. Take for example leukemia, the patient could be suffering from acute or chronic myeloid or lymphoblastic leukemia and which type of ML or LL the patient is suffering from.

Staining of slides

1) The slides (2 aspirate, 1 squash, 1 imprint and 1 blood smear) are stained with Maygrundwald-Giemsa stain for the morphological classification of hematopoietic cells.

Maygrundwald-Giemsa stain
The Romanowsky stain consists of methylene blue and its oxidation products and eosin Y or eosin B. The combined action of these dyes produces the Romanowsky effect, yielding purple colouration to the nuclei of leucocytes and neutrophil granules and pinkish colour to the erythrocytes.


2) A separate slide (usually the aspirate smear) is stained with iron stain for the classification of anaemia associated with defective haemoglobin synthesis such as thalassemia, leukaemia, myelodysplasia and sideroblastic anaemias. The identification of ringed sideroblasts is used for the diagnosis of sideroblastic anaemia and myelodysplasia.

Iron stain
Cellular iron exists in the form of ferritin or hemosiderin (storage iron) in the bone marrow. Prussian blue iron stain is for the presence of hemosiderin (storage iron) in bone marrow and stainable iron in erythroblasts (sideroblasts) and/or erythrocytes. Hemosiderin stains blue-green; nuclei of cells stains red. Iron-containing granules will be demonstrated in siderocytes and normally in many of the normoblasts (precursor of erythrocytes) of human bone marrow. Extracellular iron deposits will be demonstrated in bone marrow. This siderotic material (hemosiderin) is distinct from ferritin, which is water-soluble and not detectable by Prussian blue reaction.


Examination under the microscope

Aspirate smears are able to show RBCs, WBCs and platelets. Squashed smears will be able to show whether the bone marrow is hypocellular or hypercellular. Imprint smears will be able to show cells that are present on the surface of the bone marrow core (bone).

Types of cells one will be able to see in the smears under the microscope: blasts such as myeloblasts, promyeloblasts, monoblasts and lymphoblasts, myelocytes, promyelocytes, promonocytes, eosinophils, basophils, erythrocytes, megakaryoblasts, megakaryocytes, prolymphocytes, etc.

Malerie Goh

TG02

More answers::

To Xin Yi:
Upon consulting my mentor there, I found out that it is not possible to tell you the 'common' colours of moulds as different moulds express a different colour. Even as the mould grows, the colour may change. For e.g Aspergillus spp. may first appear white, then as it grows, turn to any shade of green, yellow, oange, brown or black, depending on its species. Thus, colour of the mould is used as a guide and not a confirmatory test for identification of mould.

To Leslie:
Err.. yes. In that fungus lab, only fungus identification is carried out. Should there be any bacteria present, they will report 'bacterial growth' and let the doctor decide whether a seond specimen should be sent for bacterial culture etc. The fungus lab will not undergo the entire process of identifying what type of bacteria it is.

As for what plate to use, it depends on what type of specimen it is. For example:
- Nails, skin scrapping: SDC + AA plates are used.
- Fluids, brain and oral cavity: SDA + SDC tubes are used.
- Tissue: SDA + SDC + BHIA + BHIB + CG tubes are used.

To Farhana:
Pseudohyphae are long branching filamentous cells of fungus, that are separated by cross-walls known as septa. These are normall(Look at the long strands of cells. watch out for the cross-walls)


http://www.uni-duesseldorf.de/WWW/MathNat/mikrobio/ernst/interest-Dateien/intere2.gif

Blastoconidia are yeast cells, thus they are not found in moulds. These cells reproduce by budding. Budding means that the parent blastoconidia separates to form a daughter cell.


http://bugs.bio.usyd.edu.au/Mycology/images/Topics/StructureFunction/buddingYeastCells.jpg

To Jean:
OOpps. should have defined the terms. These terms actually describes the gram-morphology of the organism.
- GNB: Gram-negative bacilli
- GPC: Gram-positive cocci


All righty. Hope all questions are answered in the best possible way =)!

Hope it helped!

-Li Ping-
TG 02

Answers::

To Ka Hang and Yvonne (your second question):
The germ tube test is carried out by:
1. Inoculate the yeast into the horse serum.
2. Incubate the serum at 35 – 37oC for 2 - 3 hours.

So, no subculture is needed. We can just do a wet mount directly from the tube. No gram stain will be needed.
3. Do a wet mount using the serum.
4. Observe the slide under the microscope.

To Yvonne (first question):
API is kit that consists of a combination of biochemical test used for identification of organisms. There are different types of API avaliable. For example, for identification of Candida, the API 20C AUX is used. So in conclusion, different types of API kit are used to identify different types of organisms.

To Andika:
To differentiate these Candida species, the same type of API method will be used. But this API intepretation is different from the API method for bacteria.
- For yeast, a change of turbidity will be noted.
- For bacteria, a change in colour will be noted.

To Xin Yi:
About the colour of moulds, i've gotta check it out cos' different moulds have different colours. But i'll find the more common colours for you.

About the cornmeal agar, there is 1% Tween 80 added as a basal medium. This will help to encourage the growth of chlamydospores.


All righty. Hope that I've helped make understanding easier =)!!

-Li Ping-
TG o2

Week 11: Microbiology

Another 5 weeks passed and it's my turn to post again. Anyway, 5 weeks gone by and I'm shifted to another lab. Fungus lab. A little F.Y.I: In Singapore, the lab must have a special license to operate a fungus lab. Also, fungus cultivation is also a slow process (with incubation time of at least 2 weeks), with low profits and high risks. So many labs may not have a fungus lab, preferring to send their specimens out to a lab with a fungus lab.

Cultivation of fungus is like, as my mentor says, maintaining a garden. Fungi are pretty interesting to look at under the microscope. Instead of the normal GNB and GPC, we can see beautiful formations of conidia and hyphae shaped like flowers.



As different fungi has different morphology, it is possible to observe the fungi under the microscope, note the formation of conidia and hyphae, then identify what type of fungi it is. So unlike bacteria identification, very little biochemical tests are carried out.

Filamentous fungi (moulds) can be differentiated by observing:
- Colour of the mould
- Formation of the spores
- Growth rate
- Temperature differences - some mould grow better at higher temperatures, while others may
grow better at lower temperatures.

Candida spp. are another type of fungi known as yeasts. There are 2 strains that can be differentiated from the other strains. They are Candida albicans and Candida dubliniensis. These 2 strains can be differentiated from the other strains using these 3 tests:
- Germ tube test
- Use of cornmeal agar
- Urea test

Germ tube test: Candida albicans and Candida dubliniensis will produce germ tubes when inoculated into horse serum. These tubes supposedly contributes to the pathogenecity of the organism



The long tubes at the end of cells are germ tubes. (http://upload.wikimedia.org/wikipedia/commons/1/12/C_albicans_germ_tubes.jpg)

Cornmeal agar: It is a medium made up of cornmeal and agar with added Tween 80. After inoculating the organism on the agar, incubate the agar for 18 - 24 hours. Then observe under microscope. These following features should be noted:
-Pseudohyphae
- Blastoconidia
- Chlamydospores

Candida albicans and Candida dubliniensis will have these 3 features while the other Candida spp. will not have chlamydospores. Chlamydospores are lollipop-like cells.


Chlamydospores (http://www.forestresearch.gov.uk/website/FCPicLib.nsf/LUImagesByFilename/43887.jpg/$FILE/43887.jpg)

Urea test: It is basically the same as the one used for bacterial identification. Candida albicans and Candida dubliniensis are urease positive while the other Candida spp. are urease negative. Positive test = pink agar.

To differentiate between Candida albicans and Candida dubliniensis: use API method.

To differentiate the other Candida spp.: use API method.


The medium and agar used in the fungus lab are also different. Instead of the conventional blood agar and macConkey agar, other types of agar are used. I have summarised their abbreviations, functions into the table below. Hope it helps make understanding better.


* Click on picture for bigger view. Sorry for inconvenience =).

So, this is the end of my stint at the fungus lab. Next week, it'll be to the anaerobic lab, where i'll be using this interesting-looking incubator thing.

Hope my post have been coherent enough for understanding!

11th week gone. 9 more weeks to go =).

-Li Ping-
TG o2