Week 14..

Hellooooo.. this is already the 14th weeks think everyone should already get use to working life.. another 6 more weeks till the end everyone jia you! anyway this week is my turn to blog again have a nice day reading..

I have been doing the control assay since the last post and I had gotten the result. You might be wondering why I took so long to get just one result. Due to some mistakes make and repeating of the same experiment a few times in order to confirm the results. Anyway some of you asked me about showing the graph during my last post. Now i shall show you some of the graphs that i had plotted.

Graph A Graph B

Graph C Graph D

Different gain are shown in the graphs. The higher the gain value, the higher the reading due to the higher sensitivity of the machine(Tecan plate reader). From the raw data graph, the curves for positive control show increasing reading over time, while the negative controls are quite constant over time. After getting the raw data, i will plot them into ratio(which mean positive control/negative control). From there, i can decide which gain value and which concentration of cells will give me better stability and better reading.

From graph B and D, gain 40 will give a stability for 2 hours and gain 50 and 60 will give stability for 4 hours.

My next experiment will be on the standard inhibitor.. This is to find out how long it will take for the bacteria cells to be inhibited.

Brief steps:

1. Inoculate and take OD reading of S.aureus.

2. Serial dilute and dilute S.aureus culture.

3. Prepare 96-well plate, incubate overnight.

4. Prepare ampicillin of different concentration.

5 Add ampicillin into the 96-well plate.

6. Prepare Resazurin.

7. Add Resazurin into 96-well plate.

8. Take fluorescence reading at 1 hour interval.

Have a nice day reading!!

Justina

0605950E

TG01

Biochemistry Lab
Tan Zhao Rong. Tgo1.

Blood gas analyses are performed to evaluate the respiratory function of patients namely the acid-base status (pH), the oxygenation (pO2) and the ventilation status (pCO2). This blood test is specifically performed on blood from an artery and thus is somewhat more uncomfortable and difficult to perform compared to venipuncture.

The reference range for blood pH should range between 7.35 and 7.45. The pH status would indicate if the patient is acidemic or alkalimic.

The reference range for pO2 ranges between 75 – 100mmHg. A low o2 indicates that the patient is not respiring properly (hypoxemic). At a pO2 level of less than 26mmHg, the patient is at risk of death and must be oxygenated immediately as insufficient oxygen is transferred to the vital organs such as brain and heart.

The reference range for pCO2 should range between 35.0 – 45.0 mmHg. Any abnormal pH status would indicate a respiratory problem as the pCO2 in arterial blood is determined entirely by ventilation. A high pCO2 indicates under-ventilation, where respiratory acidosis occurs and acidity of blood rises. A low pCO2 would indicate hyperventilation where there is increased alveolar respiration.

ABL 825 flex blood gas module is the instrument responsible for carrying out blood gas analyses. The specimen type required would be 1 – 3 ml of arterial blood in heparinized syringes.

Procedure:
1. It is necessary to mix a blood sample before introducing it to the system/instrument so as to ensure homogeneity. RBC is to be evenly distributed and mixed within the syringe so as to prevent machine from aspirating a plasma phase or packed RBCs from the specimen. This may result in inaccuracy in results produced.
2. Check for any clots by injecting the first few parts of the specimen into a waste container.
3. Ensure that the analyzer is in READY mode and scan the barcode. (Barcodes would be labeled on the patient’s request form and specimen once it arrive.)
4. The inlet is a section of the instrument in which the analyzer aspirates specimens. Therefore, the next step would be to place the syringe tip firmly in the inlet and press START.
5. Press Aspirate to start the measurement.
6. Remove the sample when prompted by the analyzer.

When specimen volume is too low, it would be difficult for the machine to aspirate fully from the syringe as some parts may remain at the tip of the syringe. So as to fully utilize the whole specimen, the specimen can be transferred to a capillary tube before inserting into the inlet. A capillary tube can hold up to 200ul of blood specimen and the minimal requirement for a blood gas analysis would be to fill up to three quarter of the capillary tube (150ul).


Factors that may cause result to be inaccurate:

- Frothy samples or visible air bubbles would affect the pO2 and pCO2 level in the syringe causing inaccuracy in results.

- Sample not properly anti-coagulated are rejected as clots can cause the instrument to break down. Therefore, blood samples in normal unheparinized syringes are not accepted.

- Sample should not be left in room temperature for more than 30 minutes.

- Sample should be delivered in ice so as to slow the metabolic processes which causes inaccuracy. Delays in analysis (without chilling) may result in inaccurately low

Cytopathology

Cytopathology

Hi guys!
How are you all doing? It’s already halfway through SIP!! :)

For the first 2 entries, they are all about Histology, but now, I’m going to talk about Cytology :)
For us, we have to take turns to be attached to Cytopathology Lab for a month. So I’m now in my third week in Cytology, and let me share about one component of it, ie. Erythrocyte lysis. And btw, Cytotechicians basically spend long hours (the whole day) screening slides, and they will take turns to be stationed in the processing room to prepare and process smears.

Cytology is different from Histology in that specimens sent to Cytology are fluid-based and are to be screen for pre-malignancy. Specimens sent to Histology are tissue specimens that consist of tumors, polyps (basically,already malignant).

The most common specimens received in Cytology are:
Non-gynaecological:
Sputum, Aspirate, CSF and urine

Gynaecological:
Cervical smear, vaginal smear

Some of these specimens may be blood-stained, and this blood contamination may obscure microscopic examination of cellular materials. (They only want to look at the specific cells for the tissue type and the inflammatory cells/WBCs)

So, we use hemolytic agent to help us.
1. Clarke’s solution, or
2. 0.9% Saline solution (equivalent to tap water)

Clarke’s solution
· Contains acetic acid and 95% alcohol
· Smears are placed in Clarke’s solution for 15-30 mins, until the RBCs are lysed (judging by the colour of the smear)
· Smears are then rinsed in water before fixing in 95% alcohol

0.9% Saline solution
· Smears are air-dried before placing in 0.9% Saline solution( or tap water)
· Smears are then fixed in 95% alcohol

Non blood-stained smears are fixed directly in 95% alcohol.



Ting Ying Chee
TG01

Urine Full Examination

Urine FEME

YAY WE SURVIVED HALF OF OUR SIP! Haha great job guys! :)

Anyway like what Lesile mentioned in his post, for the 3 of us in Raffles we are permanently stationed in a section each. I’m stationed at the urine section! Hence today I will share with you guys about Urine FEME.

Urine FEME stands for Urine Full Examination(FE) Microscopy Examination(ME).
As the name implies, there is 2 processes to perform upon receiving the urine samples.

I will share with guys what is FE this post and contiune with ME on my next post yeah:)

Full Examination
The 1st process to perform upon receiving a urine sample is urine full examination. This process uses urine dip stick to test for the content of the urine.

For my lab, we uses Combur-test strips and Miditron Junior II to obtain the results.

Test for:

S.G (Specific Gravity):
o Purpose: Check the amount of substances in urine, which shows us how well our kidneys balance the amount of water in urine.
o Range: 1.005-1.030

pH:
o Purpose: Measure how acidic or alkaline the urine is.
o Range: pH 4.5-8
o In our lab, we use the pH to determine the type of crystals observed in urine.
(Will be further evaluated under ME)

Pro (Protein):
o Purpose: Detect for kidney diseases; measures albumin level in urine
o Range: neg, trace, 1+, 2+, 3+
o If pro is > 1+, we need to look out for cast in ME
o Fever and pregnancy may cause protein in urine.

Leu (Leucocytes):
o Purpose: Detect for UTI
o Range: neg, trace, 1+, 2+, 3+
o If leu is > trace, we need to look out for WBCs in ME for confirmatory.

Nit (Nitrate):
o Purpose: Detect for bacteria infectioin
o Range: neg or pos
o Even if it is pos, report only if there is present of bacteria.

Glu (Glucose):
o Purpose: To detect for diabetes
o Range: neg, trace, 1+, 2+, 3+
o If glu is > trace, do a confirmatory test with Keto-Diabur-Test 5000 urine dip sticks.

Ket (Ketones):
o Purpose: To measure whether fat is metabolized properly in our body.
o Range: neg, trace, 1+, 2+, 3+
o If ket is > trace, do a confirmatory test with Keto-Diabur-Test 5000 urine dip sticks.

Ubg (Urobilinogen):
o Purpose: Detect for hepatic dysfunction
o Range: neg, trace, 1+, 2+, 3+
o If ubg is > 2+, perform confirmatory test by repeating Combur-test strips.

Bil (Bilirubin):
o Purpose: Detect for hepatic dysfunction
o Range: neg, trace, 1+, 2+, 3+
o If leu is > 1+, perform confirmatory test with bilirubin tablet (ICTOTEST® Reagent Tablet)
(If tablet turns absorbant purple, bil is pos)

Ery (Erthrocytes):
o Purpose: Pathologically significant for several diseases
o Range: neg, trace, 1+, 2+, 3+
o If leu is > 1+, we need to look out for RBCs in ME for confirmatory.

For more details, visit
http://www.webmd.com/a-to-z-guides/urine-test
http://www.anytestkits.com/utk-urobilinogen-in-urine.htm

Hui Min
TG01