Food Sampling

haha... i am so sorry guys... looks like we are all mixed up with the dates... i shall blog now too... poor nat got so much questions to answer...

alright. back to the basics. as u all know, i am still attached to the food and water lab. so now, i shall take all of u on an excursion on how i collect my samples! (food sampling)

For our lab, we do sample collection from hotel's restaurants. To go and collect samples, we first need to prepare some items.

Ice Box with an Ice Pack - this is to lower the temperature of the food to slow down or minimise the multiplication of the microorganisms. Swab the ice box with beacoup first!

Sterile spoons - To collect samples

Sterile bag - To store samples

Marker - To label the time, date, place of collection, the name of the sample and person collecting.

Empty bag - To dispose the used spoons.


After packing all the necessary stuff, we get to sit in the company's car to the hotel!

When we reach the hotel, we proceed to find the chef in charge. They will provide us with the samples that we need to collect for testing.

When collecting samples, always use the sterile spoons. DO NOT use tongs or utensils provided by the hotel as they might have been contaminated. ALWAYS practice aseptic techniques when collecting food. DO NOT touch the food sample onto any outer surface of the sterile bag as it is already exposed to the environment. After collection, seal the sterile bag up. Write the time, nature, date of sample. Write also the restaurant's name and the name of the person who collected it. After that, store the food in the ice box. Seperate the raw food and cooked food with the ice pack.

After collecting all the samples, we are now ready to head back to the laboratory. While waiting for the driver, we get to take a look around the hotel!

Back at the laboratory! place all the food samples into the refrigirator and store until the time when the food is going to be tested!

Thats all! hope you enjoy this post! lol... see you guys in school soon!

~Jeremy~
TG01

Hi all! I'll be touching on an assay that should be familiar to all of us ---- Bradford Assay.

Bradford Protein Assay is actually a simple and accurate procedure that is used to determine the concentration of protein in solutions.

Bradford assay is a protein determination method that involves the binding of Coomassie Brillant Blue dye to proteins. The dye exists in 3 forms : cationic (red), neutral (green) and anionic (blue). The dye is usually in the protonated red cationic form. However, when the dye binds to protein, it is concverted to a stabe unprotonated blue form. It is this blue dye form that is detected at 595 nm in the assay using an ELISA plate spectrophotometer.

Usually a standard bovine serum albumin (BSA) (2mg/ml) is used to plot the standard curve that will be used to extrapolate the protein concentration of the samples. Various concentrations of the standards BSA will be prepared as follows:

Concurrently, the protein samples are also diluted, usually in a 20x dilution. Once these are prepared, the tubes are then vortex to allow the well mixing and centrifuged to bring down all the proteins.

For my experiments, both the standards and samples are pipetted into a 96-well plate in triplicates, which means that for each standard or sample, there will be 3 wells. 250ul of Coomassie Brillant Blue dye aka Bradford reagent is then added in. Take into consideration that this step is time critical because bradford reagents are light sensitive. Therefore, very often, an aluminium foil will be used to cover the 96-well plate after the addition of the reagents.

When the absorbance are given by the spectrophotometer, a standard graph of absorbance value against protein concentration is plotted. An example of the graph is shown :

With this standard curve, the protein concentration can be extrapolated.

One thing to note when plotting the graph is the R-squared value. The desired R-squared value is 1. However, it is not possible to get this value unless there are only 2 standards. Therefore, for my lab, as long as the R-squared value is greater than 0.99, the graph is accepted and the protein concentration that is extrapolated from this graph is said to be reliable and accurate. If the desired R-squared value of greater than 0.99 cannot be achieved, the assay will be carried out again.


Thats all for bradford assay! Its just sweet and simple! Hope u guys understand! Feel free to ask me any qns!

Take care and see ya all in 2 weeks time!

Charmaine
TG01

Reticulocyte Count

Hi all.
I am kinda lost with the shedule on whoose gona blog this week or what, so i'll just post something okay (: sorry to the one who ought to blog this week! HA.

Okay, i shall introduce Recticulocyte Count Test that is done in the Haematology Section;

1) Recticulocyte Count (RC) (in another words, rectic count)

Introduction: The recticulocyte count is used in the evaluation of anemia as it accurately reflects the amount of erythrocytes production taking place in the bone marrow.
Relationship btwn anaemic condition and erythrocyte production in bone marrow (in normal cases):
anaemic condition= increase in RBC production therefore increase of RC in blood
However, if the RC is not raised, it shows an indictation of impaired bone marrow function or lack of eythrocytes stimulus.

Principle: Recticulocytes are junvenile red cells. Thus they contain remnants of the ribosome and the ribonucleic acid, which are present in larger amt in the cytoplasm of the nucleated precursors from they are derived.
New Methylene Blue or Brilliant Cresyl Blue are supravital dyes that are used to measure reticulocytes.
Currently, there are 2 methods being employed:
a)Manual Method: The use of Brilliant Cresyl Blue
b) Automated method: Reticulocytes Package by Cell Dyn Ruby analyzer
We used the automated method cause its more efficient and contributes to a faster Turn Around Time (TAT).

Procedure of Automated Method

1) When using the retoculocyte reagent, verify the expiration date and store the stock reagent in the dark at room temperature
2) Label patient accession No. on to the tube of reticulocyte reagent
3) Verify that the whole blood specimen is warmed to room temperature and well mixed pior to
sampling
4) Pipette 20 uL of the blood sample into each labelled tube of reticulocyte reagent
5) Incubate the stained Reticulocyte specimens on a rotator or in a rack, after fully inverting the stained specimens 4-6 times. Inbubation must be performed according to the reagent package insert.

Note: The stained Reticulocyte specimens must incubate for at least 15mins but no more than 2 hours pior to processing on the Cell Ruby machine.

Although the process is time-efficient, there are some limitations of the procedure.

Okay im done with elaborating. Hm i shall list some definations of defined abnormalities (that we 'kinda' need to know and understand in the haema section)


Leukocytosis is an elevation of the white blood cell count (the leukocyte count) above the normal range. The normal adult human leukocyte count in peripheral blood is 4.4-10.8 x 109/L. A white blood count of 11.0 x 109/L or more suggests leukocytosis.
Leukocytosis is very common in acutely ill patients. It occurs in response to a wide variety of conditions, including viral, bacterial, fungal, or parasitic infection, cancer, hemorrhage, and exposure to certain medications or chemicals including steroids. Leukocytosis can also be the first indication of neoplastic growth of leukocytes.
For lung diseases like pneumonia,tuberculosis etc. WBC count are very inportant for the diagnosis of the disease that means leucocytosis can be seen in above mentioned diseases

Neutrophilia is a condition where a person has a high number of neutrophil granulocytes in their blood.
Neutrophils are the primary white blood cells that respond to a bacterial infection, so the most common cause of marked neutrophilia is a bacterial infection.
Neutrophils are also increased in any acute inflammation, so will be raised after a heart attack or other infarct.
As well as increasing in number, neutrophils show other changes in infection and inflammation.
A neutrophilia might also be the result of a malignancy. Chronic myelogenous leukemia(CML or chronic myeloid leukaemia) is a disease where the blood cells proliferate out of control. These cells may be neutrophils. Neutrophilia can also be caused by appendicitis.

Lymphopenia is the condition in which there exists an abnormally low number of lymphocytes in the blood.
Lymphopenia can be caused by various types of chemotherapy, such as with cytotoxic agents or immunosuppresive drugs. Some malignancies in the bone marrow will also cause lymphopenia.
A decreased number of lymphocytes (notably T cells) is present in those with AIDS. People exposed to large doses of radiation, such as those involved with Chernobyl can also exhibit a lymphopenia.
Lymphopenia may be present as part of a pancytopenia, when the total number of blood cells are reduced. This can occur in marrow failure.

Monocytosis is an increase in the number of circulating monocytes. In humans, 950/μL is regarded as at the upper limit of normal; monocyte counts above this level are regarded as monocytosis.
Monocytosis often occurs during chronic inflammation. Diseases that produce this state:
Infections: tuberculosis, brucellosis, listeriosis, subacute bacterial endocarditis, syphilis, infectious mononucleosis and other viral infections and many protozoal and rickettsial infections (e.g. kala azar, malaria, Rocky Mountain spotted fever).

Eosinophilia is the state of having a high concentration of eosinophils (eosinophil granulocytes) in the blood. The normal concentration is between 0 and 0.5 x 109 eosinophils per litre of blood. Eosinophilia can be reactive (roughly, allergic) or non reactive.
Diseases that feature eosinophilia: Hypereosinophilic syndrome, Parasitic infections, allergic disorders.
The release of interleukin 5 by T cells, mast cells and macrophages stimulates the production of eosinophils
Anemia is a deficiency of red blood cells (RBCs) and/or hemoglobin. This results in a reduced ability of blood to transfer oxygen to the tissues, causing tissue hypoxia.
The three main classes of anemia include excessive blood loss (acutely such as a hemorrhage or chronically through low-volume loss), excessive blood cell destruction (hemolysis) or deficient red blood cell production (ineffective hematopoiesis).

Basophilia is an uncommon cause of leukocytosis. Basophils are inflammatory mediators of substances such as histamine. These cells, along with similar tissue-based cells (mast cells), have receptors for IgE and participate in the degranulation of white blood cells that occurs during allergic reactions, including anaphylaxis
Causes; Infections: viral infections (varicella), chronic sinusitis Inflammatory conditions: inflammatory bowel disease, chronic airway inflammation, chronic dermatitis
Thrombocytosis is the presence of high platelet counts in the blood, and can be either reactive or primary (also termed essential and caused by a myeloproliferative disease).
High platelet counts can occur in patients with polycythemia vera (high red blood cell counts), and is an additional risk factor for complications.

p.s. the definations above are just F.Y.I okaaay.

See yall soooon! (:

Natalie
TG01

Dengue NS1 Antigen Strip

Hi Everyone! Goodness, I can't believe our SIP is ending soon. I'm already getting so used to working in a clinical lab. Heh.

Anyways, today I will be blogging about another testkit. I know I keep blogging about testkits la but at my workplace, almost everything is automated, even the pre-analytical stuff. Thus, I get very little hands-on tests to perform. Other than testkits, of course =).

Correct me if i'm wrong but I think somebody has already blogged abt the Dengue Duo Cassette which is a testkit for Dengue Serology (IgG/IgM). Soo, I will blog about the Dengue Antigen NS1. Yay.

Name of Test: Dengue Antigen Detection/ Dengue NS1 Ag Detection

Introduction:
Singapore has recently been hit with a dengue plaque with the number of cases rising steadily to about 13 000 and 19 already succumbed to this illness. Common indications include a high fever and a hematology report stating a sudden, steep drop in platelet count with or without an increased hematocrit. The patient may also have dengue antibody (IgM for first infection or Igm and IgG for secondary and subsequent infection) present in the serum. During the acute phase of dengue, when the antibody titer is too low to be detected using testkits, the NS1 antigen is used as a marker. This antigen is present from the onset of the fever and remains in the serum up to 9 days after the onset of fever.

Principle, Method and Result Interpretation:

• The method adopted in this testkit is lateral flow immunochromatography. The testkit comes with 25 test strips and a migration buffer.
• The test strip itself has membrane that was formed with a sample pad, a conjugate pad and a membrane where the result lines will be displayed.
• The sample pad is for loading the sample. The conjugate pad contains gold colloidal particles that are coated with anti-NS1 monoclonal antibodies and also gold colloidal particles coated with streptavidin.
  
• Method:
• 50 microliters of the sample and 1 drop of migration buffer is dispensed into a small glass tube.
  
• A strip is placed into the glass tube in an upright position in the correct orientation and incubated for 15 minutes.
  
• Should the Control Line not appear after 15minutes or appear faint and doubtful, re-incubate for a further 15minutes and interpret the results.
  
• If the Control line fails to appear after 30 mins of incubation in total, discard the strip and repeat the test with a new strip.
• Results:
• If present, the NS1 antigen will bind with the gold colloidal particles coated with anti-NS1 antibodies as the sample migrates up the strip (aided by the migration buffer) to the conjugate pad. As it reaches the Result membrane, a blue or purple line will appear.
• The Control line should appear as biotin from the migration buffer reacts with streptavidin to form a blue or purple line at the Result membrane.
• A positive result is indicated with 2 lines on the result membrane.
• A negative result is indicated with 1 line on the result membrane as the Control Line.
  
• This test can be used in conjuction with other laboratory data to aid in diagnosis of dengue even though it has a few limitations.
  

Clinical Significance:
In the laboratory, this test is used as a rapid test to detect the presence of NS1 antigen in serum. Previously, this was a test sent out to our referral laboratory where RT-PCR was used to determine the presence of NS1 Antigen. This test is a useful qualitative test that significantly helps in the diagnosis of dengue during this dengue plague in Singapore because it is a test that produces an accurate result quickly. This, in turn, aids in ensuring proper treatment for the patient more efficiently.

Cheers,
Sharifah
TG01

Technical Stuff

Hello Everybody! Sorry for disappearing for a long long time! Today, I'm back! I'm going to blog about plant subculturing which I did for my SIP. It's quite boring though.

In plant subculturing, small pieces of plant tissues are placed on or in a media rich in nutrients and sugar. The major media components are made up of some or all of the followings :

- Macronutrients
- Micronutrients
- Vitamins
- Amino acids or other nitrogen supplements
- Sugar(s)
- Other undefined organic elements
- Solidifying agents
- Growth regulators

Macronutrients :
Macronutrients provide the 6 major elements : nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) required for plant cell or tissue growth. Culture media should contain at least 25-60 mM inorganic nitrogen for adequate plant cell growth. Potassium is required in nitrite, sulfate or chloride form at concentration of 20-30 mM. Optimal concentration of phosphorus, magnesium, sulfur and calcium range from 1-3 mM.

Micronutrients :
Essential micronutrients for plant cell growth include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu) and molybdenum (Mo). Chelated form of iron and zinc are commonly used at minute concentration of uM. Iron maybe the most critical of all the micronutrients.

Vitamins :
Normal plants synthesize the vitamins required for their growth and development. Vitamins are required by plants as catalyze in various metabolic processes. As the plants are cultured in vitro, vitamins may become limiting factors for cell growth. The vitamins most frequently used are thiamine (B1), nicotinic acid, pyridoxine (B6) and myo-inositol. Myo-inositol although it is a carbohydrate not a vitamin, has been shown to stimulate growth. Therefore it is commonly included in many vitamin stock solution at concentration of 50-5000 mg/L.

Amino acids or other nitrogen supplements :
Although culture cells are capable of synthesizing all of the required amino acids, the addition of amino acids may be used to further stimulate cell growth. Amino acids provide plant cells with an immediately available source of nitrogen which generally can be taken up by cells more rapidly than inorganic nitrogen. The most common source of inorganic nitrogen are casein hydrolysate, L-glutamine, L-asparagine and adenine.

Sugar(s) :
Carbohydrates must be supplemented to the culture medium as few cell lines are fully autotropic i.e., capable of supplying their own carbohydrate needs by CO2 assimilation during photosynthesis. Carbon and energy source of plant cell culture media is sucrose. Glucose and fructose maybe substitute in some cases, glucose being as effective as sucrose and fructose being somewhat less effective. The concentration of sucrose normally ranges between 2-3%.

Other undefined organic elements :
Addition of a wide variety of organic extracts to culture media often results in favourable tissue response. Supplements include protein hydrolysate, coconut water, yeast extracts, malt extracts, ground banana, orange and tomato juice. However, undefined organic supplements should be only be used as a last resort. The addition of activated charcoal (AC) to culture media may have beneficial effect. The effect of AC is generally attributed to one of the three factor : absorption of inhibitory compounds, absorption of growth regulators and darkening of the medium.

Solidifying agents :
Agar is the most commonly used gelling agent for preparing semi-solid and solid plant tissue culture medium. First, when agar is mixed with water, it forms a gel that elts at approximately 60-100 deg C and solidify at 45 deg C; thus, agar gel are stable at all incubation temperatures. Another geling agent commonly used is Gelrite. This is a product of bacterial fermentation and should be used at 1.25 - 2.5 g/L, resulting in a clear gel which aids in the detection of contamination.

Growth regulators :
Four broad classes of growth regulators are important in plant tissue culture; the auxins, cytokinins, gibberellins and abscisic acid. Both auxin and cytokinin are usually added to culture media in order to obtain morphogenesis, although the ratio of hormones required is not universally the same. The type of morphogenesis that occurs in a planbt tissue culture largely depends upon the ratio and concentration of auxin and cytokinin present. Root initiation of plantlets, embryogenesis and callus initiation all generally occur when the ratio of auxin to cytokinin is high, whereas shoot proliferation occurs when the ratio is low.

An example of a general media receipe :

Growth regulator : MS (Murashige and Skoog) 6.45g
Vitamin : Myo-inositol 30mL
Organic elements: No name 30mL
Sugar(s): Sucrose 45g
pH 5.8 -5.81
Activated charcoal 1g

Top up to 3L with distilled water

And thats the end of my boring entry this time! Feel Free to ask any questions and sry if I bore u too much! muahahaha...

Royston Tan
0503289A
TG01

Cell lines and MTT assay

Eloooooooooooo. This week i am going to share with you guys about the cell lines that i am using for my project. For all the cell lines, i had to perform MTT assay aka cell proliferation assay. I will further explain what MTT assay is later in the post.

Firstly lets talk about the cell lines. For my project, I am using 8 human cancer cell lines and they are:

1. A431 (skin cancer)
2. HepG2 (liver cancer)
3. Chang Liver (liver cancer)
4. A427 (lung cancer)
5. CRL-2119 (pancreas cancer)
6. MDA-MB-231 (breast cancer)
7. CCL-244 (colon cancer)
8. CCL-220.1 (colon cancer)

To learn the full profile of the cancer cell lines and also the difference between the same cancer (eg. the 2 types of colon cancer), go to http://www.atcc.org/.

Based on the recommended medium (from atcc.org) ,

A431 is grown in DMEM (Dulbecco's modified Eagle's medium)

HepG2, Chang liver and A427 are grown in MEM (minimal essential media)

CRL-2119 and MDA-MB-231 are grown in a 1:1 mixture of DMEM and Ham's F12 medium

CCL-244 and CCL-220.1 are grown in ATCC-formulated RPMI-1640 Medium

Now I will talk about MTT assay.

MTT assay is a laboratory test and a standard colourmetric assay for measuring cellular proliferation. The assay measures the changes in colour and it is used to determine cytotoxicity of potential medicinal agents such as drugs.

Method to perform MTT assay(assay is done in 4 days)

1. Discard spent media from culture flask into waste bottle
2. Wash with 10mL PBS and discard
3. Add 2ml of trypsin and incubate for 2- 3 minutes
4. After cells are detached, add 8mL of media to deactivate the trypsin and mix well
5. Transfer 10mL of cell suspension into a 50mL centrifuge tube
6. Centrifuge the cell suspension at 1500 rpm for 3 minutes
7. Discard supernatant and resuspend cell pellet while washing with 10mL PBS
8. Centrifuge at 1500 rpm for 3 minutes and discard supernatant
9. Resuspend the cells in 5mL-10mL of fresh medium
10. Transfer 50mL of cell suspension into an eppendorf tube and add 50mL of trypan blue
11. Add 20uL of the stained cells to a hemocytometer and perform a cell count
12. Prepare cells to obtain a final no. of 1 X 10^4 cells / 100uL by dilution if necessary
13. Seed 1 X 10^4 cells into each well of the 96 well plate 9 (groups of 5)
14. Incubate overnight at 370C, 5% CO2 incubator
15. Next day, add 1uL of drug* into each well
16. Incubate overnight at 370C, 5% CO2 incubator
17. Next day, add 25uL of MTT reagent into each well
18. Incubate for 2 hours at 370C, 5% CO2 incubator
19. After 2 hours, add 100uL of cell lysis buffer into each well
20. Incubate overnight at 370C, 5% CO2 incubator
21. Next day, obtain the results from multiplate reader (absorbance at 570nm)

*Drug used is cis-Platinum(II) diammine dichloride of different concentrations

The yellow MTT reagent is reduced to purple formazan in the mitochondria of living cells. Then a solubilization solution (cell lysis buffer) is added to dissolve the insoluble formazan product into a coloured solution. The reduction process of formazan takes place only when mitochondrial reductase enzymes are active and therefore conversion is directly related to the number of viable cells. When the amount of purple formazan produced by cells treated with an agent(drug) is compared to the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of the cells can be deduced through the production of a dose response curve.

Ill end here for now.

Najib Bin Hamid (0503217B)