Week 14:3rd Campus Discussion Week

Name: Nurdyana Binte Abdul Rahman


Test Name: Stool for ova, amoeba & cyst test.


Hello everyone. Hope you guys are coping well. I have observed this test done at the Routine Section. The ova and parasites test is performed to identify intestinal parasites and their eggs, which are called ova, or cysts in patients with symptoms of gastrointestinal infection. Patients may have no symptoms or may not even experience diarrhea, blod in the sool, and other gastrointestinal distress. Identification of a particular parasite indicates the cause of the patient's disease and determines the medication needed to treat it.

Saline wet mount would be the best for detection of helminth eggs and larvae and it is especially good for motile protozoan cysts, which appear refractile. When cysts have been found in saline mount, they should be observed in Lugol iodine stained preparation which will reveal nuclear structure and te presence of glycogen. The nuclei stain a brownish colour and can be easily stained. For an accurate diagnosis, a concentrated method is advised so as to increase the probability of finding the parasites in faecal samples.


Para-pak spin con is a commercial kit for fecal parasite concentration of eggs, larva and protozoa. The picture below is para-pak spin con.






Picture taken from http://www.mdeur.com/products/972200.htm

Sample:


Feces should be collected directly into a clean container. It must not be contaminated with urine, water, or other materials.



Reagents:


-10% buffered solution
-Saline
-Lugol Iodine Solution

Procedure:
1. Stool(0.5g) was emusified with 10ml of saline in a plastic tube
2. 4 drops of CON-Trate Reagent A which helps to enhance the breakdown of fecal aggregrates and mucus therefore freeing parasites.
3. Plastic tube was capped and contents were mixed thoroughly.
4. The mixture were then filtered using the para-pak filter into the centrifuge tubes provided.
5. The filtered mixture were span at 2000rpm for 10 minutes.
6. The supernatant were removed.
7. And the sediment were resuspended.
8. Wet mount were performed on the sediment.
9. Then slide were examined microscopically for parasite.


The senior medical technologies will be observing the slide. Presence of bacteria and other microorganism in the stool would indicate patient is normal, thus no ova, amoeba or cyst seen. Presence of ova, amoeba or cyst would indicate an abnormal result. In addition is a sign of parasitic infestation.

Week 14: 3rd Campus Discussion Session

Name: Hardina Bte Hamzah

Topic: Cytogenetics

Title: Amniotic Fluid Set-up

Amniotic fluid is a clear, slightly yellowish liquid that surrounds the unborn baby (fetus) during pregnancy. It is contained in the amniotic sac. The fluid protects the fetus from injury and helps to regulate the temperature of the fetus. It allows the baby to move around so that the muscles and bones develop properly. It also helps the digestive and respiratory system to develop as the baby swallows and inhales and exhales it from his lungs. In addition to various enzymes, proteins, hormones, and other substances, the amniotic fluid contains cells shed by the fetus. These cells contain genetic information that can be used to diagnose chromosomal disorders. The fluid is sent to cytogenetics lab so that the cells can grow and be analyzed.

To collect the amniotic fluid, the obstetrician will insert a very fine needle through the woman's abdomen into the uterus and amniotic sac and withdraws approximately 20ml of amniotic fluid for testing. A local anesthesia will be given to the patient. The obstetrician uses ultrasound images to guide needle placement and collect the sample, thereby minimizing the risk of fetal injury and the need for repeated needle insertions.

Before starting the set-up, the medical technologist has to ensure that the labeling on all petri dishes and tubes is done correctly. The set-up must be done in the Biological Safety Cabinet (BSC) as all specimens must be processed in a sterile environment.

One conical test tube and one slant tissue culture tube are used for each specimen. Before transferring the amniotic fluid from the syringe to the tubes, invert the syringe to ensure that there is adequate mixing of the amniotic fluid. After mixing, equal amount of amniotic fluid is transferred to each tube. The tubes will then undergo centrifugation at 1200 rpm for 10 minutes. The buckets in the centrifuge must be closed before centrifugation starts.

Each specimen will have a record sheet where the medical technologist will record down the volume and appearance of fluid, size and appearance of pellet and the presence of RBCs after centrifugation are done.

A sterile coverslip will be placed in each petri dish using a pair of sterile forceps. The dishes will then be divided into 2 sets. One of the set will have the culture tube. Each set will use a different kind of media. The first set with the culture tube will use complete Chang media and the second set will use the complete Bio-AMF-2 media. Both media consists of antibiotics to prevent any bacteria from growing and contaminating the whole culture. The supernatant will be aspirated out leaving around 0.5ml in the tubes. The Chang media will be added into the culture tube and the Bio-AMF-2 media will be added to the conical tube. The amount of media to be added is depending to the number of dishes used. One dish needs 0.5 ml of media. For example, if the first set consists of 2 dishes and 1 culture tube, 1.5ml is needed to be added into the tube.
After adding the media, the pellet will be gently re-suspended and the cell mixture will be transferred onto the coverslip in the dish. The cell mixture must be confined to the coverslip. A drop of cell mixture will be placed from the conical flask to the last dish of that set. This will act as a backup. 0.5ml of cell mixture will be left in the culture tube. The culture tube will also acts as a backup.

The first set of dishes and culture tube will be placed on one tray and the other set on a separate tray. The two trays will be placed in two separate incubators with independent electrical circuits and gas sources. The cultures will be left inside the incubators and on day 3, the medical technologist will check for cells attachment. Other than that, 1.5ml of media will be added to the dishes and tube. If there is no cell attachment, media will not be added and put on hold for another day. On day 5, the media will be changed and be replaced with a fresh media. After enough cells are grown, the cells will then proceed to tissue culture.

week 14

Subject title: Clinical chem/ Haem
Name of test: G6PD screening test

Principle:

• G6PD test carried out in our lab is only a QUALITATIVE and SCREENING test
  
• G6PD is an enzyme which catalyzes the reduction of NADP+ to NADPH
  
• NADPH keeps glutathione in its reduced form


• Reduced glutathione or GSH protects red blood cells from oxidant damage and hence prevents the lysis of RBCs


• The reagent used in this test contains NADP


• Test principle is as follows:

G6PD (this is supposed to be on top of the arrow in the equation..but i juz can't move it)

Glucose-6-P + NADP+ ----->Gluconate-6-P + NADPH+H+

• The NADP in the reagent will be converted to NADPH if G6PD is present
  
• NADPH produced in the reaction will fluoresce under long-wave UV-light
  
• If there is G6PD deficiency or total absence of it, no fluorescence will be observed
  

Materials:

1. Filter paper

2. Hitachi cup

3. G6PD reagent

4. UV light

5. Incubator

6. Patient's sample in EDTA tube

Method:

1. Pipette 100uL of reagent solution into the hitachi cup which has been labelled with the patient's lab no

2.Add about 2.5uL of patient's sample into the same cup

3. Mix well and pipette 5uL onto the filter paper(1st spot)

4. After 5 mins, pipette another 5uL onto the filter paper(2nd spot) and wait for another 5 mins to pipette again 5uL of patient's sample(3rd spot)

5. Then, put the filter paper into the incubator

6. After 5 mins, observe the filter paper under long-wave UV light

ok the filter paper will look something like this, u would have three spots of blood which are made in 5 mins interval by pipetting 5ul of the patient's sample

Results:

1. Fluorescence indicates presence of G6PD

• usually the 1st spot of blood would not fluoresce well, the 2nd and 3rd ones will

2. Absence of fluorescence indicates the absence of G6PD

Note:

• Deficient cases will be followed up with G6PD quantitative test which is a send-out test


• Negative control is done before our first sample of the day

Nur Farhana Binte Ramlan

0604834B

13 th week

hello guys...

for this entry, i will blog on one of the test that is carried out in microbiogy section.

the test is STOOL CULTURE

Principle Analysis
Acute infectious diarrhoea is caused by a number of different agents: bacteria, viruses and protozoa. Each bacterial agent of infectious diarrhoea has unique pathogenic mechanism that cause a specific symptoms. These symptoms and an adequate patient history are clues that will help the physician catergorise the patient's disease. Antimicorbial therapy can alter the host's normal protective microbiota and thus predispose the patient to bacterail overgrowth that result in diarrhoeal disease caused by organism such as Clostridium difficile, staphylococcus aureus, Candida spp. and Pseudomonas aeruginosa.

For our lab, we routinely cultures stools only for the presence of Salmonella, Shingela and Vibro spp. because of their role as causative agents of bacterial diarrhoea and other enteric diseases.

Organism such as Aeromonas spp., Plesiomonas shigelloides and Yersinia spp. are identified only wen they are present in pure or heavy growth or upon request.

Specimen
stool

• collect the specimen in a sterile, dry, leak-proof container
• select portion containing pus, blood or mucus for culture
• 1 to 2 g of sample is sufficient

Reagents
media

• Alkaline Peptone Broth, AP
• MacConkey Agar, MAC
• Selenite-F Broth, SF
• Thiosulphate-Citrate-Bile salts-Sucrose Agar, TCBS
• TSA with 5% Sheep Blood, BA
• XLD

supplies

• Sterile cotton swabs
• Applicator sticks
• Inoculating loop/straight wire

storage requirements

• store all media (agar plates and broth) at 2 to 8 degree celcius
• leave the media at room temperature overnight before use

Procedures
primary inoculation

• note the appearance of the stools i.e watery, loose, bloody, semi-formed and form
• inoculate stools according to Table 1
• subculture selective broth after 18 to 24 hours of incubation as in Table 2

Culture examination
After incubation, examine all primary and subculture plates for the growth of enteric pathogens.

For Salmonella/Shigella/Vibrio cholerae screenig, examine all the primary and subculture plates for the growth of Salmonella, Shigella and V. cholerae.

For the Vibrio cholerae screening, examine all the primary and subculture plates for the growth of V. cholerae.


Reporting of results


dats all...
i hope you guys learnt something from my posting.
thanks for reading..

sutiana
TG01
0604651J

12th week

Ever since i got back from cytology, i have been at the shaving station in histo. Shaving is the process of removing excess wax and exposing the tissues in the embedded blocks. This will provide a smooth-sailing sectioning of the tissues. Shaving is done using the microtome machine. The difference between sectioning and shaving is, sectioning is done at 4-5 microns while shaving is done at 20 microns.

In addition, the person at the shaving station will be responsible for placing the tissue blocks which require decalcification into RDO. RDO is actually an acid which will decalcify the tissues. I have actually come across tissues which i cannot shave at all because it is too hard. For tissues of that nature, it will have to be placed in RDO even before it is shaved. For other tissues which do not require decalcification, they are placed in a detergent after shaving. This detergent will soften the tissue surface to the perfect level to which that it will be easier to section it. These blocks are placed in the detergent for five minutes and then washed under running water. If the blocks are placed in the detergent longer than five minutes, the staining of the sections on the slides will be compromised. Once, it was left in there for more than 5 minutes, blobs of stains were seen on the slides. This is perhaps due to chemical interactions. The detergent which has penetrated into the tissues will interact with the chemicals from the H&E stains.

There are many things to look out for during shaving. Firstly, score lines. Score lines are lines that appear on the surface of the tissue blocks. These lines are caused by the blunt blades. When score lines are observed, the blades must be removed immediately. Then, the blocks will be shaved again until the lines have disappeared. It is very important to ensure that after shaving a block which requires decalcification, the blade is changed to shave the next block. This is because, obviously, the blade will be blunt after shaving the hard tissue from the block which requires decalcification. If the blunt blade is used to shave a tissue block containing small tissues, it is hard to remove the score lines without taking the risk of losing the tissue after continuous shaving. Thirdly, removal of sutures and staples. Sutures are what seemed to be black threads used during an operation. Amazingly, sutures and staples are used by the surgeons during the operation for the orientation of the organs. Orientation of the organs aid in the trimming of the organs by the pathologists. Sometimes, i believe, unknowingly, when the pathologists trim the tissues into the cassettes, sutures or staples are hidden in tissues. The presence of the sutures and staples will apparently remain unknown throughout tissue processing as it does not distrupt the processing. It will usually be found during embedding and most of the time, during shaving. Which is pretty unpleasant. Not only will the staple or sutures cause the blade on the microtome to be instantly blunt, it will also require much digging and pulling out the staples and sutures from the hardened wax block. Fourthly, tissue blocks containing certain breast tissues are not required to be shaved. This is because they are exceptionally thin. Finally, i have to ensure that the wax in the tissue blocks are completely hardened before it can be shaved. This is to prevent the risk of the tissues dropping off from the block. When that happens, the tissue will have to be sent for re-blocking.

I realize that embedding affects shaving a lot. This is because, at times, the tissues are not on the same level in the wax. This is probably due to the tissues not being pressed down towards the mould during embedding. When the tissues are not on the same level, much shaving needs to be done to expose every part of the tissue. When this happens, the risk of losing parts of the tissues increases.


Nurathirah
Tg01
0606561I

11th Week of SIP

Name: Hardina Hamzah

Topic: Microbiology

Title: Processing of Urine Specimens

Urine culture is commonly done to determine whether the patient has urinary tract infection. A urinary tract infection is a bacterial infection that affects any part of the urinary tract. When bacteria get into the bladder or kidney and multiply in the urine, they cause a UTI. Enterobactericeae species such as, E.coli, Kleibsiella pneunomiae, Proteus mirabilis and Pseudomonas aeruginosa, cause most of the urinary tract infection.

Retrieved from: www.pharmacy-and-drugs.com

There are two types of media used and there are TSA with sheep blood agar and MacConkey agar. TSA (Trypticase Soy Agar) with sheep blood agar is useful for the cultivation of a wide variety of fastidious microorganisms. TSA is a general purpose media produced via enzymatic digestion of soybean meal and casein and TSA is frequently the base media of other agar plate type. In this case, it is the base media for blood agar plate. MacConkey agar is a culture medium to grow Gram-negative bacteria and stain them for lactose fermentation. It contain bile salts (to inhibit Gram-positve bacteria), crsytal violet dye (also inhibit Gram-negative bacteria), neutral red dye (to stain microbes fermenting lactose), lactose and peptone.

TSA with Sheep Blood Agar

Retrieved from: www.madsci.org

MacConkey Agar

Retrieved from: www.madsci.org

The urine specimen need to be processed immediately as if prolong, the results may not be accurate. The urine is mixed well before inoculating it onto agar plate. 1ml calibrated disposable loop is used and the loop will be immersed into the urine specimen bottle. The sample will be inoculated onto Blood agar plate first and then MacConkey agar plate by making a straight vertical line down the centre of plate and will be followed by series of close perpendicular streaks through the original line. The plates will then be incubated at 35°C aerobically overnight. At the same time, microscopy cell count of the fresh urine specimen has to be done. The urine sample will be transferred to the notch on the slide chamber of KOVA GLASSTIC SLIDE 10 with grids. The urine sample will be transferred by using the capillary tube and the urine sample will be drawn into the chamber resulting in a homogenous suspension of sediment. The cells to be counted are epithelial cells, white blood cells and red blood cells. When there are a lot of white blood cells (eg. >100 cells) that are counted; this can indicate that the patient may be having urinary tract infection.

KOVA GlASSTIC SLIDE

Retrieved from: http://www.cenmed.com/productDetail.asp?productid=17291&catID=&category=5617&mainCat=&cat=5305

After office hours or during weekends/public holidays or even there is a delay in transporting urine specimen, the dipslide will be used. The dipslide has three types of media which are Cysteine Lactose Electrolyte Deficient (CLED) agar, MacConkey agar and E.coli agar. CLED agar is a valuable non-inhibitory growth medium used in the isolation and differentiation of urinary organisms. Being electrolyte deficient, it prevents the swarming of Proteus species. Lactose fermenters produce yellow colonies on CLED agar; non-lactose fermenters appear blue. It has a pH of approximately 7.3. The staff nurse will dip the dipslide into the container containing patient’s urine and then seal the dipslide into the bottle. The staff in the laboratory will help to incubate overnight. One disadvantage of using this method is that there will be no urine microscopy cell count.

Uricult Trio Dip Slide

Retrieved from: http://www.oriondiagnostica.com/files/odextra/Clinical%20Microbiology%20brochure/782-03GB_Uricult.pdf

The cultures and dipslides which are made on the previous day will be read on the next day. Usually when there is a mixed growth of bateria (different types of organisms growing), it is considered insignificant and will be recorded as negative. However, when there is predominant growth of only one type of bacteria, it is considered as significant and will be recorded as positive. Enterobacteriaceae species can be identified easily by seeing the morphology of the colonies and to confirm the species, indole test can be done. For example, when E.coli is suspected to grow, indole test is done on the spot to confirm. A small filter paper is used and a drop of indole is placed on the filter paper. Then, the filter paper is rubbed against one of the colonies. When the filter paper turns blue, it indicates positive and it is proven that it is an E.coli. Then the culture will be sent for further test which is sensitivity testing. However, when there is a doubt on the identity of the organism, the organism will sent for identification and sensitivity test.