Histopathology/haematology

Hello to all friends,

i was arranged to work in histopathology for the first 2 week, then 2 week in haematology. Now i'm in processing section.

But first let me tell you the Quality Assurance Programme “here”.

- Royal College of Pathologist Of Austrialasia
- External Quality Assurance Services (Bio-Rad)
- College of Pathologist Survery (general chemistry/immunology/transfusion medicine/Dignostic Virology/Haematology/General)

Unacceptable specimen criteria:- unlabeled specimen
- mislabeled/misidentified
- unidentified specimen
- specimen submitted in improper container
- specimens not accompanied by a cytology accession forms as appropriate
- incorrect/soiled accession form
- insufficient quantity of specimen
- empty container
- improper storage/ no clinical history

Note: the submitting physician/nursing station will be notified and will be expected to submit a fresh, labeled specimen. If not, the nurse need to personally identify the specimen.


First week: histology

1) In histo lab, documentation is very important. They must perform and maintain records which required by accreditation agencies (pre-analytical QC):

- run control slide on immunochemistry
- stain quality
- maintenance records/instrument maintenance
- calibration records
- records on type and number of tests done everyday, then evaluate every 6 months(draw bar graph & file)
- records of SOPs (not online. I understand some lab are online SOPs)
- duties of technologist
In short, they record down everything they do on paper and file it.

2) Screening and reporting of gynecological specimen- pathologist maintain work logs

3) Review of abnormal cases – identify areas for continuing education/seeking the opinion of outside consultant/documentation of all reviews is essential for quality assurance monitoring

4) Rescreening of negative cases

5) Cytology-histology correlation and clinical follow up- compare pre-malignant & malignant reports/helpful in directing further patient management

6) Retrospective reviews – IQA/efforts is made to minimize bias when reviewing cases

7) Measures of screening performance – correction & prevention measures

8) Proficiency testing and continuing medical education – EQA

9) Variability in practice - automation


Procedures from the start to the end
When the specimen arrives in the lab, the technologist will sign against the name of the patient, check whether the nurses send the correct specimens (should be in 10% formalin) or else they would call to the wards and check. Specimens are arranged to non-gynae/ gynae/ urgent.

Step2: tissue processing/ scribing
Tissues are either being cut/processed by technician(mostly like small specimen like lump/tonsil/EOS drop) or pathologist. Pathologist normally are in charge of handling bigger specimen which required specific orientation that the doctor want to observe( e.g. what is the degree of spreading of tumor cells in the breast).

Tissues are then transferred to cassette. Do u still remember the 12 steps processing? Let me remind you:

1 &2 steps: formalin
3step: 70% alcohol
4step: 95% alcohol
5&6&7steps: 100% absolute alcohol
8&9&10steps: xylene
11&12steps: hot liquid paraffin wax

After the tissue are embedded into metal mould and sectioned. Some of the hospital lab have different method in sectioning. My lab is as following steps:

1) place the cell blocks onto the block holder clamp. Ensure that the block is perpendicular to the microtome blade.
2) A blunt knife is used to shave off the excess wax from the block to expose the specimen. If the t/s containing unsuspected mineral deposits, the block are placed into decalcifying solution for surface decalcification. If the specimen is skin, the block are placed into softener (ya! the one we used to wash our clothes with—make it softer)
3) Change the blade to new sharp blade
4) Transfer the blocks to a cold plate
5) Refit the block to the clamp
6) Sectioned the block @ 4-5 microns by rotating the microtome handle in the clockwise/anti-clockwise (depends on the machine) to produce thinner sections. The number of microns used also depend on the pathologist.
7) Lift the sections onto the alcoholic water to spread the sections (unfold)
8) Transfer the sections onto the warm water bath (45 degree) and use a clean glass slide to “fish” out the sections


These slides are then arranged into a rack (facing one direction) and placed into an oven @ 75 degree for 10mins.

When the time is up, the rack are placed into a containing container of the auto-stainer. (staining methods will be explained later)

Interpretation: histological slides are examined under a microscope by a pathologist and then he/she will report his/her findings. Pathologist will compare results among themselves to give a best interpretation.

H&E staining is a very common staining procedures. I am sure that everyone should know so I’m not going through it. Beside H&E stain, special stains is another methods to identify glucogen/carbohydrates/minerals.

Most common special stains:
- periodic Acid Schiff’s (PAS)
- Periodic Acid Schiff Diastase
- Alcian blue
- Congo red
- For other pls check this website: www.hoslink.com


Frozen lab is normally located next to a operating room. Pathologist will examine the t/s while the surgery is still taking place.

Reasons:
1) If a tumor appears to have metastasized, the pathologist and the surgeon will decide whether there is any point in continuing the operation.
2) If a tumor has been resected but it is unclear whether the surgical margin is free of tumor, an intraoperative consultation is requested to asses the need to make a further resection for clear margins.
3) In a sentinel node procedure, a sentinel node containing tumor t/s prompts a further lymph node dissection, while a benign node will be avoided.
4) Rapid examination of a lesion might help to identify the possible cause of a patient symptoms.

Steps:
1) When the specimen arrives, it will be immediately be assigned with a number which will be entered on the examination forms, and histology logbook and the time of receipt recorded.
2) A dignosis should be made and called to the operating room in 20mins
3) The pathologist will check the specimens, dissect if necessary, and will place the t/s to be sectioned onto a bed of OCT compound on a crystat chuck. Liquid nitrogen is used to froze the chuck.
4) The specimen is then inserted into the holder arm of the cryostat, and t/s sectioned @ 5µm
5) Once the section has been cut, it can be picked up by a glass slide over it (the t/s will “melt” onto the slide due to different in temperature). Slides are labeled with accession number.
6) 1 slide for H&E staining. Another for Tol Blue staining
7) Pathologist will diagnosis after observing the slide and record on the logbook and submission sheet. Time of receipt is made.
8) Patient’s identification checked and confirmed before delivery of any verbal report to the surgeon.
9) The remaining of t/s will undergo normal histopathology
10) Report records permeantly onto patient’s records

Second week: haematology

D-Dimer test

1) Introduction

D-dimer is a protein that is released into the the circulation during the process of fibrin clot breakdown. D-dimer represents an area of cross-linked fibrin degradation product that originated from the breaking down of the fibrin clot network during the body’s repair mechanisms. These fragments are released from the clot by the action of the enzyme plasmin. D-dimer present in circulation is used as an indication of a blood clot being formed and broken somewhere in the body.

Blood Activation


Thus it is possible to use D-dimer as a test for many medical conditions and complication. In normal people, there is a very low background level of D-dimer. Abnormal levels (elevated levels) of D-dimer can be formed in patients with Deep Vein Thrombosis (DVT), pulmonary Embolism (PE) and disseminated Intravascular Coagulation (DIC).

2) Principle

The latex particles provided in the D-D1® Test are coated with mouse anti-human D-dimer monoclonal antibodies. Test samples containing D-dimers when mixed with the latex particles suspension make the particles agglutinate. At a predetermined concentration of D-dimers that the D-D1® Test is designed for, the agglutination of the latex particles produces macroscopic clumps that can visualized by the naked eye.

3) Specimen

Blood in sodium citrated tube are centrifuge for 3 mins @ 1500rpm. Plasma is needed


4) Proceduce



The 5&6week: Processing section

I heard from the rest that different hospitals have different way of processing the specimen. For example, pei shan’s lab processing section is automated while mine is manual check.

In my lab, the processing section is where the patients specimen from various wards, clinic, A/E and other departments will reach the lab via pneumatic system. This is also the place where all the datas of patients and tests ordered are keyed into the computer before dispatching the specimens to the various sections where the tests are performed. Thus Processing is the most important section among all sections.

Let me explain the proceduce in details

Firstly, the canister carrying specimens will travel from the wards, clinic, A/E and other department through the pneumatic system to the lab. The lab have three automated lines to receive and send out results. It also have three manual lines to send results or rejected out the code of the desired wards. E.g. code 3300 is for ward 33

The specimens will come with their requested forms, and these forms are to be sorted into awaiting/urgent and non-waiting. Time has to be zapped on awaiting/urgent/A&E/blood gas/seminal analysis/blood bank forms/ add-in tests. Priority is given to the above forms.

In addition, forms for bacteriological test, histopathological tests and external tests have to be placed at certain in-tray as they will not be run in the general lab. These tests are only available in other labs in SGH/KKH/TTSH.

Next I learned to do pasting of labels onto the tubes. The most common labels are biochem, cell dyn, ESR, Hba1c, blood gas, RA, INR, Axsym, TIBC, immuno, Stool. The tubes come in different colour too( pink cap-EDTA Blue-sodium citrate Grey-fluoride Dark Blue-zinc Yellow-plain).

Labeling are important because it ensure that the samples are the correct patients and the test ordered must match. Plain tube are centrifuged at 3500rpm for 5 mins while the rest are sent to other sections. Rejection part is e same as histo(refer back to the top)


ps: sorry if it's too long

cheers,
elaine

REPLY TO QUESTIONS...

This entry is to answer questions from Kang Ting and Andre... It's easier to clarify here.

Kang Ting:
eh... i only saw it once. Only remembered that the background is pink (d/t lysed RBC), and the presence of parasites is identify by tiny blue granule-like pigment found in the WBC. As there are 4 major species and various stages of a malaria parasite infection, the morphology varies. It is hard to describe them now because i haven't really see all yet. Maybe when i get to be stationed in Haematology again, I'll put up another post to explain this further.

Andre:
Normally the patient has to fast for at least 8 hours overnight before the blood test. Due to glycolysis, the plasma should be separated from the RBC within 60 min for an accurate measurement of GHB. If the sample is received from another hospital/clinic or it is in-housed but for some reasons (lunch break etc), a fluoride tube should be used as it contains glycotic inhibitor.

Fluoride tube: The glucose concentration is stable in whole blood (no separation of plasma from RBC) for 72 hours at room temperature.
Red/Plain tube: After separation of serum from RBC, the glucose concentration is stable for 8 hours at 25 °C and 72 hours at 4 °C.

Hence, the difference in tubes depends on the delivery of the specimens to the lab. , how fast the specimens can be processed and how long to archive the samples.


For more info, pls read: Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus


Answer to my own question: How to differentiate a male PBF from a female?
Look at the neutrophils closely. You should be able to see a "drumstick" sticking out from the side of the nucleus for the female. But for the male, no (just have to be caution of pseudodrumstick). That's the difference.



-Pei Shan-

Clinical Lab - UPDATED CONTENTS in purple

Dear all, it's my turn to share with you my learning experience over the past few weeks...

I am attached to this small yet very comprehensive lab. This lab is special, we have roll call every morning and every thursday, there will be continuous education meeting before roll call to improve on the staff theoretical knowledge e.g. how to operate machines and how to handle emergencies in blood banking. We have small sections (chemistry, hematology, blood banking, urinalysis, microbiology) within the lab itself. Every day, the staff has a different role to play (e.g. Mon and Tue I am in chemistry, Wed i am in hematology, Thu I am in urinalysis etc) except for the microbiology department which has fixed staff to carry out the routine duties. Hence every med. tech. knows how to order, run and perform almost every test panel in each department. I shall share a little of what i've learnt from the departments that i've been to.


Chemistry
We run a combination of chemistry + immunology + special chemistry (which are the uncommon tests that are not run everyday) here, using MPA (Modular Pre-Analytics), SWA (Serum Work Area) and Cobas 6000. Dont worry, they are just the names of the analyzer. There are many test panels but I would like to discuss this particular test in details:

Name of test: Glycated Hemoglobin (GHB) / Hemoglobin A1c (HbA1c) Test

Principle of test: Introduction

• It measures the glycated hemoglobin (hb), also known as HbA1c, in the blood over the past 3 months.
• Hb combines with glucose to form this stable component, HbA1c.
• Individuals with high blood glucose will have high level of HbA1c.
  
• It is used in diabetes monitoring to see how well a diabetic patient manages or controls his/her diet (glucose intake).
• The patient cannot cheat the doctor by restraining from sugary food a few days before the blood test as I've mentioned, it provides an indication of the blood glucose level over a period of 3 months (as RBCs life span is 120days).
  
• Hence, it is better test than just measuring the blood glucose level at the point of the test.
• However, when a Diabetes Monitoring (DM) panel is requested, both the GHB and GLU (glucose)/GLUF (glucose fluoride) test will be ordered by the order entry staff.
• An EDTA tube is needed for the GHB test and it must be inverted a few times (to mix the anticoagulant and the blood well) before loading into the analyser (in my case, we use Cobas 6000 here).
• Note that major air bubbles may affect the results.
  
• A heparin/plain tube is used to test for the serum level of glucose. Fluoride tubes are accepted as well.

Principle of Test:

• Turbidimetric Immunoassay-quantitative Method
• The anticoagulated whole blood specimen is hemolyzed automatically on the Cobas 6000 by Integra Hemolysing Reagent Generation 2 (which uses TTAB as the detergent to remove interference from leukocytes; but it does not lyse them)
• 1. Sample + R1 (buffer/antibody) : HbA1c reacts with anti-HbA1c Ab to form Ag-Ab complexes
• 2. Addition of R2 (buffer/polyhapten) : polyhaptens react with excess anti-HbA1c Ab to form insoluble complex which is determined turbidimetrically
• Liberated Hb is converted to a derivative which is measured bichromatically during the preincubation phase (Sample + R1)
• Final result is expressed as (HbA1c/Hb) x100 = HbA1c %

Test Result with reference range:
Normal people: 4-6% (for Cobas 6000 here, it is 4.5-6.4% due to variation of ref. range in different analysers)

Diabetic patients:

• 4 to <7% (acceptable; good control of diet)
• >9% (poorly controlled glucose level)

Clinical Interpretation:
This test is used to diagnose for diabetic patients and to monitor the diet control of these diabetes. to document and monitor the degree of glycemic control of these diabetic patients, so as to prevent any chronic complication. It is also used as a quality assuarance program to assess the quality of diabetic care (the frequency of testing) in the hospitals.

GHB reading of >7% implies that the patient is not controlling the diet well enough (high glucose intake) and his/her insulin dose/dosing interval has to be adjusted.

Usually in DM, the patient is required to do a GHB test 2-3 times a year.


Order Entry (O1)
We have a pneumatic system in 01 which bring in mainly the in-house patient specimens and their test request forms. Once I received the form, I have to tally the patient name, I/C no. with the specimen labels and order the tests as requested. 1st, the doctor name is checked and keyed into the LIS, followed by the location and the room number. If the specimen is urgent, i have to enter 'U' so that all staff know that the sample must be run asap. Any add test to a previous specimen will be keyed under comment.

Next, the test panels are entered. I almost memorised all the test panels e.g. ANP2 (Anaemia Panel 2) for testing of folate and vitamin B12. If the doc select folate, i enter FOL only. But if he chose both folate and vitamin B12, i have to enter ANP2. The same goes for Bone Metabolism Panel (BMP). When either Ca/PO4/Mg is selected, i enter those chosen but when a doc ticks all the panel, i have to order BMP instead. After all the tests are ordered, the data is saved and an accession sheet is printed automatically. The sticker is pasted on the test request form and the accession labels (barcoded) are used to label the tubes.

Part of my duties also include answering calls from the wards, calling the wards to inform them of test rejection/wrong specimen sent/extra tubes needed etc, double-checking of test request forms.

More info about O1 can be found in Sharifah's entry (cus we're in the same lab) under The Lab Freaks' Blog.


Haematology
I've learnt, though not very much, how to identify cells and count them using a DC counter. It was interesting to observe the cell morphology and especially in detecting Malaria Parasite (MPME). Maybe today we detect a patient with Malaria but 3-4 days later, a new blood sample from the same patient shows absence of parasites. This shows that the treatment is successful.

My senior could actually tell whether the PBF belongs to a male or female by just observing the cells under the microscope. Guess How? Try observing the PBF from now!


-Pei Shan, TG02-

*^_^ Enjoy your days at work~ wow, 1 month is gone!

Haematology

Hi all… I was attached to sgh haematology department and will be in coagulation lab for the 1st four weeks. I was assigned to do the routine PT and APTT test using the Sysmex CA-1500.

Principle: Both PT and APTT tests are used for the investigation of haemostatic failure. The prothrombin time (PT) tests for factors I, II, V, VII, X of the extrinsic system whereas the activated partial thromboplastin time (APTT) tests for all factors in the intrinsic system (factors I, II, V, VIII, IX, X, XI, XII). For PT, the time between the addition of tissue thromboplastin to the presence of a detectable clot is the prothrombin time; ref range 9.2-11.2s.
For APTT, the citrated plasma is incubated with APTT reagent (for the activation of contact factors) after which CaCl2 is added and the clotting time is measured; ref range: 27.0-36.1s.

Procedure:
1) Check if the received requisition form tally with the patient sample. Make sure that the level of blood is above the marker found on the tubes.
2) Stamp and label form and test tube.
3) Centrifuge the tubes for 3000rpm for 180s.
4) Put the tubes in the rack and placed into analyzer. Order test through the computer system.
5) Record results. PT<8.0s, APTT<26.0s and delta check indicate that test have to be repeated (to make sure it is not a random error).
6) Results will then be keyed into the LIS.


QC: Level I and II controls (used in sysmex CA-1500) are performed at an interval for every batch of 40 samples. Results that are out-of control may indicate that the QC is expired, analyzer has problem, reagent has problem.

here is a very simplified coagulation cascade:



When: -PT ↑
-APTT normal
Most likely is F VII deficient.

-PT ↑
-APTT ↑
F X, V, II, I deficient.

-PT normal
-APTT ↑
Most likely F XII, XI, IX, VIII deficient.

Eunice
tg02
0503245C

Serology-Immunology

Hi all,

Really sry ppl. So pai sei leh. I made a big mistake in the TPHA test thing.

1. The test cells are sensitised cells that reacts specifically to the antibody produced in response to the causative agent. It is by default brown in color.

2. After everything is added in, the solution in the wells are mixed evenly by tapping gently.

3. How the particles settle after the incubation gives you the result. (so must take care not to agitate the plate during the incubation and even before you read and verify the results.)

4. Particles concentrated in the shape of a button in the centre of the well with a smooth round outer margin is read as non reactive.

5.Definite large ring with a rough multiform outer margin and peripheral agglutination is read as reactive.

6. Particles concentrated in the shape of a compact ring with a smooth round outer margin is read as a boderline case. This is reported as "possible biological false positive result". A repeat testing in 10 days along with a FTA-antibody assay would be suggested to clarify if the patient is positive or negative.



Hope that helps in clafying some doubts =)
Sincerely,
Yeng Ting

Serology-Immunology

Hi all,

I am posted to a private clinical lab for SIP. Of which, my first 3 weeks is spent at the serology department before moving on to other departments of the laboratories.

Serology is basically a branch of immunology that deals with testing of patient's serum.

During this 2 weeks, I was mainly assigned to do the VDRL testing and Human ASOT because the other tests would require the usage of the LIS, which attachment students are not allowed to access.

This is the summary of the VDRL testing.

Principle of the test

RPR measures IgG and IgM produced in response to lipoidal material released from damaged host cells and also the lipoprotein released from Treponema pallidum. Thus, the antibodies detected are not specific for T. pallidum, which is the causative agent for syphilis.

Qualitative test
1. Using a Pasteur straw, place 100 µl of test serum into a circle of the test card.
2. Use the flatten tip of the straw to spread the serum evenly over the circle area.
3. Shake a plastic dropper containing the carbon antigen provided in the test kit to evenly mix the carbon.
4. Invert the bottle, holding it vertically to dispense a drop of the antigen. Each drop is approximately 0.4 µl.
5. Place the test card on an automatic rotator and rotate at 100 rpm (rounds per minute) for 8 minutes.
6. Read and interpret the results.

Semi-quantitative test
This test is performed if the result for the qualitative test is positive. This is to determine the antibody titre to aid the doctor in the treatment of the disease because the antibodies tend to disappear after successful treatment.To confirm the diagnosis, TPHA (T. Pallidum Haemagglutination) is performed.

The steps are the same as the qualitative test but instead, dilutions of the patient's serum of up to 1:32 is made using saline solution.

This is the Summary of TPHA testing.

Principle of the test

Gelatin particles, sensitised by purified T. Pallidum, agglutinate in the presence of antibodies against T.pallidum in human serum. A purple colored button would be developed after 1 hour of incubation at room temperature. This is compared with the positive control for interpretation of the results.

1. Label the test plate with the last 3 digits of the patient's ID and "C" for the positive control.

2.Prepare serial dilution of the patient specimen as follows:

Well no Diluent Specimen Test cells

1 190 10 75 for all wells
2 100 100 (carried over)
3 100 100 (carried over)
4 100 100 (carried over)
5 100 100 (carried over)
6 100 100 (carried over)


Final dilution
well 1 1 : 20
well 2 1 : 40
well 3 1 : 80
well 4 1 : 160
well 5 1 : 320
well 6 1 : 640

3. Mix the content by gently tapping the tray and incubate for 1hour at room temperature.

This is the summary of the Human ASOT

This test is basically antigen- antibody reaction of latex particles coated with stabilised streptolysin O as antigen against anti-streptolysin O (ASO) antibodies of patient's serum. This antibody is produced in response to group A Streptococci bacteial infections such as rheumatic fever or glomerulonephritis.

The method is basically the same as RPR. Instead of using a white background card, a black or dark background card is used. Instead of carbon antigen in the RPR, latex particles are used (caracterised as a milky colored solution). Instead of rotating for 8 mins in the RPR, 2 mins of rotation is needed or else it would give a false positive result.

I have also learnt how to use the Bio-Rad coda for EBV, HSV-1, HSV-2 and chlamydia IgG/IgM testing and was allowed to attend the training for the usage of their newly purchased equipment, the Bio-rad Evolis, which has an extensive list of tests it is able to perform.

The senior I was attached to also taught me how use the Serodia-Myco II test kit to test for anti-mycoplasma pneumoniae antibodies although I have yet to perform the test.

Cheers,

Yeng Ting

Question and Answers: Microbiology

Q1) What does XLD plate stands for?
Ans: Xylose lysine sodium desoxycholate (XLD) plate

Q2) What makes the Salmonella colonies black?
Ans: The Salmonella(non-glucose fermenter) produces H2S when growing which causes the colonies to turn black
Q3) How Rotavirus Quick test works?
Ans: It works by detecting the Rotavirus antigen using monoclonal antibodies (Elisa mtd). The test kit has monoclonal antibody againts the Rotavirus antigen, if the stool contains the virus a coloured pdt will be formed.


Q4) What is a MUG test?
Ans: Is by using the principle of antibody binding to the specific antigen on the Ecoli and under UV light the Fluorence compound labelled on the antibody will emit light(Ecoli positive)

Q5) What do you mean by using 3 parts of the loop?
Ans: The picture shows a inoculation loop which i have colour coded and numbered it. When doing the primary streaking use the Black part of the loop, Green part when streaking the secondary, and red part when doing the tertiary streak.(seen above)


Q6) What is a Primary, Secondary, Tertiary streak?

Ans: Refer to diagram above.

Q7) Why must the stool samples be subcultured onto the 3 different types of media (XLD plate, Campylobacter Plate, Selenite F broth)?
Ans: We want to look out for Salmonella which will grow as black colonies on the XLD plates, Campylobacter plate to look out for Campylobater jejum species(tiny grey colonies on the campy plate), so we will be able know the specific cause of the diarrhoea. The selenite culture is just for subculturing the stool sample on to a XLD plate after incubation for 1day.

Q7) Growth and identifying campylobacter.
Ans: The Campylobacter jejum species looks like pin-point grey colonies on the black background of the campy plate. a test can be done to verify the bacteria(Hippurate test). By adding the colonies into the hippurate reagent and incubate for 10mins and then add 7drops of Ninhydrin reagent and incubate for 2hrs-positive will have a purple coloured ring on the upper part of the solution.

Q8) For the quick test for rotavirus antigen identification.. are there any negative or positive controls to be done when doing it?
Ans: actually the blue band is already a control band so we do not have to run a seprate control. If the blue band does not appear, the test is not considered and the test must be redone.

Q9) For the vp3 test, how useful is it in diagnosis of microorganisms infection in the female reproductive system?
Ans:According to the manufacturer, the test kit's sensitivity is 100% and the specificity is 99%.

Q10)for UTI diagnosis,what would be done if its negative for the test, is there any further investigations that would be performed if the results turned out to be negative (not E.coli)?
Ans: It depends if the FEME count is it high if it is high we must issue a prelim report to the doctors to indicate no significant/no bacteria growth but also reflect the WBC count, so the doctor can monitor the patient's progress

Q11) What is a zig-zag line on the agar?

Ans: Refer to above diagram. the sequence of streaking is written in the picture. for the top part the streak is very close together and then when it comes close to the bottom the space is wider(but remember to maximize the space avalible at the sides. Also no note that the streaking must be done fast to prevent the urine from drying up and the streak must be consistent.

Q12)What are the main differences between MacConkey and CLED?

Ans: for CLED it is a more specialized media used for isolation, presumptive identification, for my lab we use the CLED for those overnight samples that is sent to us after working hours as if urine is not directly plated within 2hours it must be kept at 5degrees C. By using a dip slide which contains 3 types of agar: Mac, CLED, colourless base medium which is for identification of Ecoli. Mac agar inhibits gram pos bacteria which are unlikely causes of UTI thus we plate it on mac to rule out the gram pos bacteria.

Cheng Hong: Mircobiology

Subject title: Microbiology

Urine culture for diagnosis of Urinary Tract Infection(UTI)

• To culture patient’s urine sample to look for bacteria growth(mainly E.coli) which is the main cause of UTI
• Using a 1µl inoculating loop and dip just below the surface of the urine sample and draw a straight streak down the plate(both MacConkey and blood plate), then steak zig-zag lines across the agar(this is to obtain isolated colonies for identification and testing
• Incubate the plates overnight aerobically
• Obtain the plate the next day for identification of bacteria colonies
• If there is growth(pure/mix bacteria growth), look out for E.coli which morphology is white, slightly bigger than other bacteria colonies and has a very “typical smell”
• Do a indole test on the suspected E.coli colonies: if test result is green(positive), if its pink(negative)
• If the bacteria is a fermentative Ecoli the MacConkey plate should be reddish(ferment lactose) if it is not(yellow plate), we have to do a MUG test to confirm if it is a E.coli using Bacticard-Ecoli test kit which is bought commercially
• First, 1drop of rehydrating solution is added, then pick up some of the colonies using a inoculating loop and add to the test card and incubate at RT for 15mins
• Then add 1 drop of substrate and look under fluorescence light, if there is fluorescence (positive for Ecoli) vice versa
• At the same time for each patient a Urine FEME(WBC,RBC,Epithelial cell count) is also used to help in the diagnosis
• If the patient’s WBC count is high(50,000cfu/ml onwards there is a high chance of the patient being diagnosed with UTI if there is Ecoli growth on the agar
• After reporting the results to the doctors through LIS the culture is sent for verification and sensitivity testing(Antibiotic susceptibility testing)
  
  
  Stool culture for diagnosis of cause of diarrhea
• For diagnosis of diarrhea there are 3 main types of bacteria we are looking for: Shegella, Campylobacter, Salmonella
• For stool culture there are 3 types of media which is needed to be inoculated on(XLD plate, Campylobacter Plate, Selenite F broth)
• (Working behind a fume hood) Use a wooden stick to obtain some stool sample and inoculate on the XLD and Campy plates) each plate can have 2 different patient sample)-do streaking
• Use the remaining stool sample on the stick and put it into the Selenite broth
• Incubate the plates overnight(XLD, Selenite in 5% CO2 incubator and Campy plates in anaerobic jar with gas pack at 42oC-2days)
• Obtain the plates for interpretation the next day(XLD plates)
• If there is growth of black colonies(colonies are not individual, merge with neighboring colonies) we can interpret as growth of salmonella(cause of diarrhea due to ingestion of uncooked/ partially cooked meat)
• Send the plates for verification and sensitivity testing
  
  
  Rotavirus Antigen Identification
• Pick up patient’s stool sample and inoculate in to 1ml of buffer(comes with the commercial test kit)
• Gently mix the sample with the buffer
• Add 4drops of the solution from step 1 into the Rotavirus Quick test kit
• If only a blue line is seen the patient is negative for rotavirus antigen
• If a blue and red line is seen, the patient is positive for rotavirus antigen
  
  
  VP3 test
• Vp3 test is done using a vaginal swab to test for Trichomonas, Gardnerella, Candida
• The swab is placed into a tube and the excess is cut off using a scissors(remember to disinfect with isopropyl alcohol)
• 12 drops of lysis solution is added and the tube is capped and incubate it in a heating block for 10mins at 85oC
• Then add 12 drops of buffer and gently flick the tube and cap it with a filter nozzle
• Add 4 drops of substrate solution to the 7th well of the test kit and dispense the solution from the tube into the first well
• Then the PAC(test card) into the first well and place the whole kit into the Affirm VP3 machine and run the machine
• The machine will automatically move the PAC from the first well to the last well
  *those who want the contents of the individual wells please let me know
• At the end of the run if there is a blue colour on any of the respective organism it shows that the patient have the infection

*When streaking the plate always use 3different sides of the inoculation loop to make the primary, secondary and tertiary streak to be able to get isolated colonies.

*When doing biochem test or other test(eg:indole/oxidase test) use colonies grown on natural media(eg:blood agar)

If there is any question regarding the postings please comment, thanks

*Cheng Hong*