SIP- Histopathology

Hey everyone! I'm attached to Histopathology for the whole of 20 weeks. This week, i'm scheduled to do special staining for one month. Most specimens will undergo routine H&E (Haematoxylin & Eosin) stanining while some will need special stains for diagnosis.

After fishing of specimens onto slides, excess water must be drained out before placing on the hotplate for 3 minutes to melt off the wax. Then, it can be loaded into a rack for staining.

H&E staining
Principle: The purpose is to demonstrate the tissue constituents in contrasting colours. Hamatoxylin gis oxidised to haematein in the presence of an oxidising agent. Haematein is taken up by nucleic acids present in the nuclues in the presence of a mordant which are usually metallic salts that is able to chelate the haematein dye to the tissue component. Eosin stains the cytoplasm and connective tissue pink.
For routine H&E staining, the process is fully automated with the help of this machine (Leica Autostainer XL):

Extracted from:http://www.spencerscicorp.com/html/slidestain.html

This is how it works: After loading the rack into the machine, press LOAD and check that the machine is programmed to routine H&E staining. After that, the whole staining process will be carry out by the machine in this format:

1) Xylene--> 2 minutes

2) Xylene--> 2 minutes

* Steps 1 & 2- remove wax that has not melt off as wax are not water soluble.

3) Abs alcohol--> 1 minute

4) 95% alcohol--> 1 minute

5) 70% alcohol--> 1 minute

*Steps 3-5- remove xylene and rehydrate the specimens

6) Water--> 1 minute

*As haematoxylin (Hx) is aqueous based, must wash with water first

7) Hx--> 4 minutes

8) Hx--> 3 minutes

* Steps 7 & 8- stain the whole specimen 9)

9) Water--> 1/2 minute

10) 0.5% acid alcohol--> 2 dips

* Differentiation- remove excess Hx
11) Water--> 1 minute

12) Lithium carbonate--> 2 minutes

* An alkali which blue the specimen when place in water

13) Water--> 2 miuntes

* Blue in water

14) Water--> 3 minutes

15) Eosin--> 1/2 minutes

* Stains cytoplasm

16) 70% alcohol--> 1 minute

17) 95% alcohol--> 1 minute

18) 4 rounds of abs alcohol--> each for 1 minute

* Steps 16-18- dehydration as water cannot mix with xylene)

19) 3 rounds of xylene--> each for 1 minute

* Remove alcohol as depex used for mounting is xylene-based and also to raise refractive index of tissue

Results:

• Nucleur component- blue
• Cytoplasmic component- red

After the above process, the specimens will be send for mounting which is also automated.

Special staining

Principle: Some cases need special stains for diagnosis. H&E is not able to demonstrate the desired components.

Types of special stains:

• Periodic Acid Schiff


• GMS Fungus


• Reticullum ll


• Alcian Blue


• Iron


• Giemsa


• Congo Red


• Gram

Procedures:
1) Print barcode label(s) (according to biopsy number assgined to the specimen)
2) Paste lable(s) on the frosted end of the slide
3) Dewax and hydrate slide
4) Load the slide covered with special wash solution to prevent the specimen from drying up into the instument
5) Load appropriate stain kit into the reagent carousel (depending on the type of stains)
6) Select Run
* Pre-checklist will appear. Ensure all slides and reagent kit are in place. Cap of reagent kit must be open.
7) Enter number of slides and hit the Run button
The machine will now initiate the staining by reading the slide and reagent barcode labels. After the barcodes are read, information for the staining will be 'downloaded' from the NexES computer to the staining module and the run will resume. When the run has completed, click 'SIGN OFF' and remove the slides from the instument. Drain slides then load into rack and place in 95% alcohol to remove residual liquid before dehydration, clearing and mounting.

Manual Staining
Principle: The Ventana NexES machine are not able to carry out some special stains. An example is the Ziehl Neelsen staining. (I only perform this manual staining so far)

Tuberculosis Test/ Ziehl Neelsen:
Principle:To demomonstrate acid fast bacteria belonging to the genus mycobacterium responsible for causing tuberculosis. (diagnosis test for tuberculosis)

Control: Any tissue containing acid fast organism.

Reagents required:

• Commercial TB colour carbo reagent--> stains tubercle bacilli red
• Loeffler's Methylene Blue--> background staining
• 1% potassium hydroxide
• 1% acid alcohol
  
  Procedures:
  1) Dewax and bring sections to water
  2) Stain with commercial TB colour carbo reagent for 5 minutes
  3) Wash in running water
  4) Differentiate with 2 rounds of 1% acid alcohol until colourless (patient is negative), light pink (patient is positive)
  5) Wash in water
  6) Counterstain with 1% Loeffler's methylene blue for 10-15 seconds
  7) Wash in water and go to 95% alcohol to control intensity of the blue colour
  *if very blue, go to 70% alcohol (the more diluted the alcohol, the greater the differentiation)
  8) Check under microscope, if too much blue has gone off due to differentiation, repeat steps 6&7
  9) Dehydrate in absolute alcohol, clear in 3 rounds of xylene and mount
  
  Results:
• Positve- red tubercle bacilli on blue background
• Negative- sky blue

Finally, its the end of my long posting. LOL. Feel free to ask me any easy questions. Hope you guys continue to learn more from SIP! Take care.=)

June Tham
TG02
0505073G

Cytology Pictures

Centrifugers

Safety Cabinet Level 2 b

Safety Cabinet Level 2 b

Virkon solution

Autostainer

0.5% Sodium Hypochloride

Shandon reagent

Oven

Cytocentrifuge funnel, clamp, filter paper and labelled glass slides

Cytocentrifuge funnel, clamp, filter paper and labelled glass slides ready for cytocentrifugation



Disclaimer: These pictures were taken by me with permission from my supervisor; the latter has seen each picture and allowed me to use them to enhance the learning process.

Student Internship Programme - Cytology

Hey people, Desmond here. I've been attached to the cytology for 4 weeks now and have been doing non-gynaecological procedures for the first 3. In this posting, I will guide you through what is done from the time a urine specimen is recieved right until it is "reported" to the cytologist.

I will be posting pictures soon and feel free to ask any EASY questions =].

Subject title
Pathology (Cytology)

Aims
Cytology testing of Urine for malignancy

Introduction
The principle of cytology testing of urine is to differentiate the nucleus and cytoplasm of the cells to detect malignancy by comparing the Nucleus: Cytoplasm ratio (N:C ratio). The Papanicolaou staining method is used for this. It is a polychrome staining reaction (staining the cytoplasm of different cells different colours) designed to exhibit differences in cellular morphology, maturity and metabolic activity. Because intact cells in a cytological smear tend to overlap and some appear in 3D configurations, the greatest value of the Pap staining method are the resultant transparency of he cells and clear definition of nuclear detail.

Urine however has small number of cells; Cytocentrifugation is needed to pull the cells into a confined/defined region on the glass slide.

Materials
Glass slides
Depex
Xylene
Autostainer
Centrifuge
Cytocentrifuge
Cytocentrifuge funnel and clamp
10ml centrifuge tube
22mm by 22mm coverlips
Labels/Stickers
Shandon reagent
0.5% sodium hypochloride
Virkon solution

Methods
(a) Receiving Specimens (Urine)

1. Scan the time onto the patient’s form
2. Fill in the type of specimen and initials of the person receiving the specimen (e.g. DH)
3. Label the patient’s form with pre-made stickers which are chronologically numbered and bar-coded.
4. Assign a cytologist to the case and record details like case number and the cytologist assigned to the case.

(b) Processing specimen (urine)

1. Enter Biosafety Cabinet Level 2.
2. Remove the container with the patient’s urine from the biohazard bag
3. Compare IC numbers on patient form and the container
4. Label the container and a 10ml centrifuge tube with the pre-made stickers (e.g. NG1234/07)
5. Indicate on the patient’s form the following:
Volume of urine
Condition of urine (e.g. Colourless, bloody or yellowish)
Initials of the person processing the urine
Date
Number and type of stains required (in this case 2 Pap stains)
6. Gently mix the urine by overturning the container
7. Aliquot about 10ml of urine into the 10ml centrifuge tube
8. Centrifuge the urine for 10 minutes at 2000rpm.
9. Prepare 2 labelled glass slides for cytocentrifugation
10. Decant the supernatant into 0.5% Sodium Hypochloride
11. Add 6 drops of Shandon reagent to the cell pellet.
12. Mix by pipetting up and down
13. Add 3 drops of the mixture of cells and Shandon into the cytocentrifuge funnel.
14. Cytocentrifuge for 6 minutes at 800rpm
15. After cytocentrifugation, fix the glass slides in 95% alcohol.

(c) Staining

1. After fixation for 20 to 30 minutes, load the glass slides onto a staining rack.
2. Load the rack into the autostainer
3. Set the autostainer to the Papanicolaou staining programme.

(d) Mounting

1. After staining, mount the 2 glass slides.
2. Dip the glass slide into xylene
3. Add a drop of depex to the stained side of the glass slide
4. Place a coverlip on the Depex
5. Press out any air bubbles
6. Place the glass slides into an oven (45 degrees Celsius) to let the Depex solidfy (5 to 10 minutes)

(e) Microscopic observation and reporting

1. Observe under microscope to determine if there is an adequate number of cells or staining for the cytologist to detect any abnormalities/malignancy

2. Report the case including the patient’s form and glass slides to the cytologist to detect abnormalities (malignancy)

Results
(Reference)
Nuclei – Blue
Acidophillic cells – Red
Basophilic cells – Blue-green
Erythrocytes – Orange-red

(Urine tested)
Nucleus appears very large when compared to cytoplasm. There are multinucleated cells and the nuclei appear to be intensively stained blue-green.

Discussion

The patient’s Cells are malignant. There is nuclear enlargement without an increase in the overall size of the cell, giving a decreased cytoplasm to nuclear ratio. There is also hyperchromasia due to increased amounts of DNA. The nuclear outline appears irregular and there is a variation in size and shape. Moreover, nucleoli increase in size and number. Abnormal cell division results in multinucleation. Finally, there is uneven distribution and variation in size and chromatin particles.











Desmond Heng Chih Pheng


0503179D


TG02

Long-awaited answers!

....

Despite my despairing pleas, everyone didn't heed them and -still- posed tough questions for me! sigh.. and here goes,



Phui Yuen: hahaha yeah countless PBFs done, but there's still room for improvement! regarding your qns 'under what disease/clinical presentation is ESR done? or that ESR is just a general routine test done?', ESR isn't considered a general routine test but a miscellaneous(single) test ordered separately by the consultant himself/herself. ESR, as stated, is a nonspecific screening test whose results do not confirm a diagnosis of diseases. for eg, a patient can have a normal ESR readings and still have a problem. so yeah, basically it's up to the consultant to decide if he/she wants the test done to aid in diagnosis but that will be more towards clinical aspect.



Azhar: 'Since you said that the erythrocyte sedimentation process can be affected by a number of factors, is there any special equipment that is used to minimise these factors?'

hm actually there isn't any special equipment. perhaps the special and most important one is the ESR stand to support the tube in a vertical position for a more reliable and accurate reading. if you had noticed in the factors that i've stated, most of them would largely lean towards human variation(errors). also, the ESR stand is secured at the middle of the table and not near the ends where the alignment may not be equal and certainly not near any centrifuges.



Eugene & Nisha: hahaha. 'what's hairy cells?' they are cells with 'hairs'. ok lame :P the image is taken from a previous control slide but the presence of hairy cells in the blood basically shows that the patient is suffering from hairy cell leukemia(hcl), and hcl is classified under chronic lymphoid leukemia and a b-cell disease. the abnormal b-cell will have hairlike cytoplasmic projections on its surface. i dare not go into details for i'm not too sure myself!



Doreen: yeah my lab uses hematek2000 stainer but occasionally, manual staining using leishman stain. that's during time constraints. as for FBC report reflecting low platelet count, the follow-up actions will be to check for fibrin clot in the sample using wooden applicator sticks, any presence of giant platelets or platelet clumps in the blood film diagnosis which will be screened by the senior med techs and after which, they can proceed by doing manual differential count when necessary and report significant RBC and platelet findings.



Yeng Ting: to your first qns 'Rouleaux formation is the stacking of erythrocytes? Is the settling of erythrocytes equal to rouleaux formation?' yes, rouleaux formation is the stacking of RBCs atop one another. in ESR, the basic principle of the test is for the RBCs to form rouleaux and as the complex gets 'heavier', it generally settles down into what we call the packed cell volume. to your second qns 'In what ways can the ESR results be affected?' let me put this simply with a patient with sickle cell anemia as an example, sickle cells unlike normal RBCs are shaped like crescents. these sickle cells will still settle down but unable to form a typical rouleaux like normal RBCs probably with 'gaps' between them. so, going back to the principle of ESR, results will be affected by the structural variations of cells and in this case, give a decreased value.



Ying Ying: hahaha. i didn't see this coming. 'Why perpendicular and not vertical?' define perpendicular and it has the meaning of being vertical and upright in the sense that the 90-degree angle is created by the table surface and the ESR tube. so, simply put it this way, perpendicular in this case equals vertical!



Eunice: !!! 'Why are there 2 reference ranges for females?' the reference ranges listed were obtained locally based on results of healthy individuals. there being 2 ranges for females divided at about 50 years are probably due to normal physiology of females like menopause etc. there are significant changes of the mean result of females at <50>51 years during the tabulation of data and so the reference ranges were divided as so for females.



Lizzie: really? your place received over a thousand samples for ESR?! wow. hahaha.

ESR here are done manually. whether it is done before or after FBC totally depends on the amount of blood sample received. ideally, it would be done before FBC due to the waiting time of an hour but if there aren't enough blood in the sample to run both FBC and ESR, running FBC will be the priority.



Chaur Lee: yeah there certainly will be interference of results after 60 minutes as RBCs will still continue to settle down beyond the time frame resulting in false high readings. here, the count-down timer is a med tech's best friend. the reading is taken plus/minus 1 minute of the 60 minutes when the alarm sounds, so there is virtually a remote possibility that these results are not used. however, it's human to err so you wouldn't want to discount human errors if it happens? and yup, variations in RBC affect ESR results but in this case, ESR being a miscellaneous test is ordered by the consultant so maybe it's fair to say that we do our jobs by performing the test and report the results. :P



Wing Fat: 'What is platelet satellitism?' i must admit this is a new term to me when i saw the control slide and took a pic to share with you guys! how could you do this to me.. but yeah after some research(thanks ah), platelet satellitism is characterized by platelets forming around polymorphonuclear leucocytes(neutrophils) and are seen in blood films stained by Wright's stain prepared from EDTA-anticoagulated blood samples and not seen in other anticoagulants like sodium citrate, heparin etc. wouldn't go into details as this is only what i've learnt! thanks ah wing fat.



Jiaxin: hahaha. i suppose if the company is paying for your living, it is a must to learn to do everything. us as interns, if the supervisor deems you fit as to run patient samples, you are being treated like one of them and will be good to help around to your abilities and learn at the same time. so, the answer to your question is DO EVERYTHING!



Juexiu: for panic alarms such as excessively abnormal levels of platelets, haemoglobin and total white, we have to inform the wards immediately. while for general abnormal results from tests, we report the results and include comments with it.



Ci Liang: yeah, variations in RBC sizes affect the ESR results. i asked a senior med tech and she told me ESR for children were done mostly done in the western countries and it had been done here in the past, but not at present. but i've also found a reference range for children of 0-10mm/hr developed at Baptist Memorial Health Care Corporation, Memphis, Tennessee. hopefully it helps. :D



Peishan: this question got me thinking for quite a while, and i actually set up an experiment today just for you! it is definitely important to mix the ESR tube thoroughly before setting the test up to achieve a state of homogeneity between the components of blood. if blood is not mixed well, RBCs will settle at the bottom and form 2 clear distinct layers of plasma and RBCs. from what i've observed from my experiment, setting up the ESR test on blood that was not mixed(tube 1) will therefore allow the plasma region to be catapulted right to the top of the tube in place of homogenized blood from the control(normal ESR/tube 2). from the start, in tube 1, it can be observed that it isn't a fair test due to the segregation of plasma and RBC layer giving a reading of about 8mm following by the RBC layer that is unevenly spread while in tube 2, the reading is at the zero mark. after 60 minutes, the readings which i recorded were 85mm/hr and 32mm/hr in tubes 1 and 2 respectively. this clearly shows that the tube without mixing gives a false high result. after discussing on the findings with a senior med tech, it can be said that in the tube 1, some areas of the red cell layer had already settled down which MAY had formed rouleaux. with this uneven distribution of weight in the vertical ESR tube, say at the centre, it may force the RBCs further down along with gravitational pull which will give a high reading come the end of 60 minutes. these are of course, based on my observations and discussion, i really hope you did understand what i'm trying to bring. perhaps mr alvin poh[if he does read] can explain why?



Joan: both primary and secondary controls are commercial controls, given by the manufacturer of the automation. in the morning, the 3 levels of control (low/medium/high) is run in the machine before running patient samples to ensure that results fall within the manufacturer's given reference ranges. running secondary control also utilises the commercial controls but the difference is that only 1 of the 3 levels are used at the given time intervals. this is part of internal quality control and it also functions to keep patients' results in check within the reference ranges and that results are quality-assured.



Sharifah: hahaha in the lab, the ESR station is secluded in the middle of a single table. only materials involved in ESR are placed there, namely ESR stand, ESR tubes, timer, record book etc. the station is usually left alone after setting the ESR tests and no other materials known to cause vibrations are placed there. the only vibrations then, can only be due to people treating the table like a drum! but trust me, why play at the station? it's no fun! :P

AHHHHHHHHH! and trust me, answering questions are no fun! but of course, end of the day, i hope i did help in easing those lingering doubts that you once had and learnt something from my experience!

case closed! *wide grins :D* till my next post.. have fun everyone!

Peek-tures

Mixing of blood to prevent clotting

Patient with chronic granulocytic leukaemia

(Note the huge amounts of total white cells

as well as different stages of differentiating cells)

Hairy cells

Platelet satellitism
(Clumps of platelets adhering to neutrophils)

ESR station

Images released with the permission of company supervisor

- Alex

Student Internship Programme (SIP) HAEM

Howdy my coursemates!

I've been scheduled to the routine lab of the haematology department for the first 4 weeks (not first6weeks :D). It's not named the routine lab for nothing and surely there is always going to be a general routine flowchart to follow. So, here goes (a brief one)!

1) Starting up the automation at the start of operating hours (8am), daily maintenance check, run quality control (QC)
2) Run commercial controls a.k.a primary controls (low/medium/high) during startup and secondary controls (either one of low/medium/high) at specific intervals, say 10am, 12noon, 2pm
3) Collect patient samples and test request forms at the reception and run various tests
4) File unverify automatch results
5) Delta check prompt? (it means a significant difference in current and previous results spotted by the LIS in any tests done in the same individual patient)
6) Screen blood films (BF)
7) Full blood count (FBC) result tally with BF diagnosis?
8) Enter and verify results in LIS

In case you're wondering what sort of tests are done in the routine lab, there are full blood count, miscellaneous tests like

- Retics
- Haemoglobin (Hb)
- Malarial parasite (MP)
- Erythrocyte sedimentation rate (ESR)
- Platelet
- White blood cell (WBC)
- HbH test etc..

The test that I've chosen to elaborate will be the ESR.

Name of Test: Erythrocyte Sedimentation Rate (ESR)

Principle of Test: ESR test measures the settling of erythrocytes in human plasma over a specific time period, usually 60 minutes. The reported numerical value is derived from measuring the distance (millimeters) from the bottom of the surface meniscus to the top of the erythrocyte sediment in a column of anticoagulated blood that has remained perpendicular in a special-purpose pipette (solely for ESR usage) for 60 minutes.

A typical sigmoid curve resulting from the descent of the plasma-red interface plotted against time distinguishes three phases of ESR. The lag phase (initial portion of curve) reflects the period where individual erythrocytes form rouleaux. During the decantation phase (second portion), the plasma-red interface falls more rapidly. In the final phase, the cell aggregate pile up on the bottom of the tube but may not always be evident within the 60 minutes time frame. The final ESR test result, read at 60 minutes, will therefore include varying contributions from the three phases.

The erythrocyte sedimentation process can be affected by a number of factors such as
1) incorrect amount of blood for dilution, 2) air bubbles in ESR tube, 3) inadequate mixing of sample before and after dilution, 4) failure to ensure vertical alignment of tubes and 5) vibration.

Conditions such as variations in erythrocyte shape for eg, sickle-cell formation or acanthocytosis (mixed erythrocyte shapes) will inhibit the ability of erythrocytes forming rouleaux and hence, ESR results will be affected.

Test Results with reference range:
Male - 1-10 mm/hr
Female (up to age 50 years) - 3-15 mm/hr
Female (age 51 years and above) - 3-20 mm/hr

Clinical interpretation: ESR is a nonspecific test used to detect inflammatory, neoplastic, infectious and necrotic processes which are associated with a modification of the plasma protein (primarily fibrinogen, globulins and albumin). It is not diagnostic for any particular disease or injury. It is a fairly reliable indicator of the course of disease and therefore can be used to monitor disease therapy.

I hope I've put up a fair recollection of what's happening around me and share a thing or two with everyone.

Have mercy please. Don't kill me with all the questions! :/

- Alex Tg02 0503222B

Haematology laboratory

Second week of SIP

Hello everyone… I was posted to haematology department for the whole of 20 weeks. This week I was scheduled to thalassaemia lab and will be there for the next two weeks. Well, I had learnt quite a lot at the lab dealing with the blood samples that were sent in for Hb electrophoresis. Firstly, I will collect patient’s blood samples at the reception sent to them by telelift, transporter or pneumatic tubes. After which, I will do labeling on the samples and check that the patient’s data tally with the request form. Next, key the specimen no. into LIS.

Test performed: Cellulose acetate alkaline electrophoresis

Type of specimen: a minimum of 3.0ml of EDTA blood
EDTA preferred but heparin is acceptable.
Reject clotted blood and lyse specimen except for clotted cord blood (very difficult to get the blood)

Purpose: it is used as the initial procedure to screen for hemoglobin variants. The technique is sensitive to blood samples throughout the human life span (from newborns, cord blood, to old age)

Principle: Hb has a net negative charge at pH8.6 and move in an electrical filed towards the anode (positive) due to the variation in the amino acid content off different Hb, the net charge of each Hb types varies and this will determine their rate of mobility. Various supporting media such potato starch, paper and polyacrylamide gel have been used in electrophoresis. Cellulose acetate is used because it is easily available and provides a sharp resolution of the Hb bands in a short time; allow cleaning, densitometric quantitiation and permanent storage of the transparent film.

Procedures:

1) Pipette 200microlitires of blood to tubes and wash 2x
2) Pipette 25microlitres of washed packed red cells into the tube.
3) Add 150microlitres of hemolystate reagent and allow standing for 20 minutes.
4) Label the plate with specimen no. using water proof marker and place in carrying rack. ( the other side of the plate is the cellulose acetate)
5) Slowly, lower the rack into heme buffer and soak for 5 minutes. ( it must be done slowly to prevent any air bubbles)
6) Pipette 10microlitres of the patient’s blood and hemolystate into each sample well. Control is pipette into the first well. Control used is ESFA. E and S are abnormal variants. F and A represent fetal and adult respectively.
7) Prime the applicator by depressing gently into samples well 1 to 2 times and blot on a piece of filter paper.
8) Remove the wetted cellulose acetate and blot dry
9) Place the plate in the aligning base with cellulose acetate side up
10) Transfer the applicator to the aligning base
11) Apply samples to plate (on the cellulose acetate) by the contacting the tips (“teeth”) of the applicator to the plate and hold for 5 seconds.
12) Place the plate in electrophoresis chamber with cellulose acetate side down
13) Electrophoresis the plate for 25 minutes at 350volts


Staining of bands

1) The plate is stained in ponceau S stain for 5 minutes
2) Destain in 3 successive washes of 5% acetic acid (2 minutes for each wash)

Evaluation of bands

For qualitative evaluation, the haemoglobin is inspected visually for the presence of abnormal haeomglobin bands. EFSA control provides a marker for band identification

For quantative evaluation, the relative percentage of each haemoglobin band is determined by scanning the plate in the densitometer using 525nm filter.

Record results

taken from http://www.dokkyomed.ac.jp/dep-k/cli-path/a-super/h25.html

Test performed: sickling test

Purpose: used to demo the sickling phenomenon in test samples and a confirmatory test following alkaline cellulose acetate electrophoresis


Procedure:

1) 25microlitres of reagent is added to 5microlitres of EDTA blood used the pipette tip to mix to a coin shaped
2) cover with cover glass and left dry
3) seal with grease and incubate at 37degrees for about 2 hours
4) examine microscopically
5) record results


Quality control: include a negative and positive control to check that reagents are okay.

That’s all… have fun for SIP yea :)

kai lin

0503211E

tg02

Microbiology Laboratory

I was posted to microbiology laboratory for 2 week. Read and understood the laboratory safety manual and general protocol.

Was sent to the processing bench for the first 2 days. I learnt how to clocked in the various samples that was sent to the microbiology laboratory to verify the time that the samples were received. I was taught how to differentiate the various nature of the specimen so as to allocate them to the various benches for tests. The blood bottles was first incubated in an incubator for to check for sign of mircoorganism growing in the bottle. The type of test is denoted by the colour of the cap. Blue represent aerobic culture, golden represent anaerobic culture while red represent fungal culture. Urine specimen regardless of catheter or mid-stream urine was first culture in a blood and macConkey spilt-well agar. It is noted that streaking must be done on blood agar first before macConkey so as to avoid carry-over effect from macConkey agar to blood. MacConkey agar will encourage growth of gram-negative bacteria. Specimen that were not urine or blood were labeled as miscellenous item. All swab of superficial wound will be culture on blood and macConkey agar, while addition culture on PEA plate to be carried out if swab of site is below waist. PEA agar encourage growth of gram-positive bacteria. Type of culture to be carried out depend on appearance of stool. If it is a normal stool, culture will be carried out on macConeky, Salmonella-shigella agar and selenite broth. Addition culture will be carried out on watery stool. They are TSA agar with 5% sheep agar, thiosulphate Citrate Bile Salt sucrose agar and alkanline peptone water. These agar will help to detect the presence of Vibro which are usually associated with watery stool.

Supervisor then allocated me to the microscopy bench on the third day to observe the various procedure. I was taught to differentiate the appearance for the respiratory specimen. It may be mucoid, plurelent, watery or blood-stain. Depending on what type of stain the doctor request, the medical techologist will then process the request. Usually gram staining is carried out. Slide is stained with crystal violet for one minute and then washed. Slide is then stained with iodine for 2 minutes follow by washing and rapid decolourising by 100% acetone. Slide is lastly counter-stained with safranin for 1 mintues and then read under microscope. I was taught to identify blastoconidia and mycellium under the microscope. If acid-fast stain is request, it must be read under an immuno-fluroscent microscope in the dark room to identify mycobacellium.

I was then allocated to the urine bench on the fourth and fifth day to observe interpretration of the urine culture that was cultured on the blood and macConkey spilt-well plate. Colour and appearance of the culture will be noted to aid in the interpretattion. Usuaaly, if there is green colour on the blood agar culture, it implies culture is alpha hemolytic, and if there is pink colour culture on the macConkey agair, it implies culture is a lactose fermenter. Addition information such as whther culure is in swamps will aids in the identifcation.If culture contain less than 10^5 and is not a pure culture, no further testing will be carried out as this is usually due to contamination. Addition tests that are carried out may be radID one procedure, staph grouping kit, indole test etc.

Yeo Ching Wei
0503288C
TG02