Week 10 SIP/MP

Pictures for the vitro vents requested.

Hello all, after reading the comments in my last post, I have decided to explain the different components of the media and I hope that everyone will understand better why the different components must be added.

MEDIA

Media components

The composition of the plant media is important for the plant media to grow properly. Plant tissue is usually made up of the following components: macronutrients, micronutrient, vitamins, amino acid or other nitrogen supplements, sugar(s), other undefined organic supplements, solidifying agents or support systems and growth regulators. For this post, I will be touching on macronutrients, micronutrients.

The media formulation that is used- Murashige and Skoog formulation contains the macronutrients and micronutrients. Macronutrients provide 6 major elements needed for plant growth: Nitrogen, Phosphorous, Potassium, Calcium, Magnesium and Sulfur. 25-60 mM of inoraganic nitrogen is needed in a culture for adequate plant growth. Potassium is needed for cell growth of plant species, most media have potassium in the nitrate, sulfate or choride form at concentration of 20-30 mM. The rest of the elements: Phosphorous, Magnesium, Sulfur and Calcium are included at concentrations ranging from 1-3 mM. Higher concentrations of these nutrients may be needed if dificiency of other nutrients are present. Micronutrients are elements such as Iron, Manganese, Zinc, Boron, Copper and Molybdenum. Chelated form of iron and zinc are commonly used, with Iron being very important. Iron citrate maybe used in culture media but the compounds are had to dissolve and usually precipitate out after media is done. Murashige and Skoog uses ethylene diaminetetraacetic acid-iron chelate to overcome this problem. Sodium and Chlorine are also used in some medium but are not essential for growth. Iron and Molybdenum are added at concentrations of 1 microM, Zinc at 5-30 microM, Manganese at 20-90 microM, Boron at 25-100 microM and Copper at 0.1 microM

Week 9 MP - Culturing of Stenotrophomonas maltophilia

Hi everyone...! I have changed my MP due to some unforseen circumstances (which I shall not elaborate further) and it will be somehow similar to Benjamin's MP.

MP title: Development of 2D-Zymogram protocols to detect and identify Stenotrophomonas maltophilia periplasmic proteases and compare their activity at 37°C vs 28°C.

Quick facts on Stenotrophomonas maltophilia (S.maltophilia)

• Previously known as Pseudomonas maltophilia or Xanthomonas maltophilia
• Sole member of the genus, Stenotrophomonas
• Gram-negative bacillus (rod-shaped) which is gram-stained pink
• Aerobic (require oxygen to grow and survive)
• Non-fermentative
• Motile by means of flagella
• Found widely in environment and hospitals
• Infects immunocompromised individuals especially

Why study S.maltophilia ?

• Important nosocomial pathogen
• Highly antibiotics resistant
• Little is known of its virulence factors, genetic structure and pathogenicity
• Ageing population
• Increased use of surgical equipments

Culturing of S.maltophilia

Principle: To allow the growth of S.maltophilia on LB agar plate and to obtain single, isolated colonies of the bacterium.

Materials

• S.maltophilia isolates (in vials, frozen state)
• Disposable inoculating loops
• LB agar
• 37°C incubator
• Biosafety cabinet 2 (BSC 2)
• 70% ethanol
• Marker pen
• Biohazard bag
• Appropriate personal protection equipments (e.g. Lab coat, covered shoes and gloves)

Methods

1. Set up BSC 2.
2. Dry LB agar in 37°C incubator.
3. Swab vials (containing S.maltophilia isolates), inoculating loops and LB agar with 70% ethanol.
4. Place the necessary materials into the BSC 2 and arrange them orderly.
5. Label the LB agar plate.
6. Streak on the LB agar plate with an inoculating loop (with S.maltophilia).
7. Dispose used inoculating loops into biohazard bag.
8. Swab the LB agar plate with 70% ethanol before incubating at 37°C overnight.
9. Observe for single, isolated colonies.

Explanation of methods

1) To provide a sterile environment for culturing to prevent any contamination. To protect the user from S.maltophilia (safety reason).

2) To prevent the formation of water droplets from condensation as the LB agar are stored in the fridge.

3) Aseptic techniques

4) To facilitate workflow within the BSC 2.

5) To facilitate identification of S.maltophilia isolate.

6) To transfer S.maltophilia onto the LB agar plate.

7) Used inoculating loops are considered biohazard wastes and should be disposed in biohazard bag and autoclave later.

8) To disinfect the outer surface of LB agar plate with 70% ethanol before incubation. Incubation at 37°C overnight allows growth of S.maltophilia optimally.

9) This shows that streaking was done properly (recall streaking techniques from Basic Microbiology) and the colonies will be used for subsequent experiment such as inoculation.

That's all for this week!

Tan Han Yang

0606190G

TG01

Week 8 SIP - Chloroform Shock

Hello everyone. It is the 8th week of SIP/MP and its my turn to blog again. This week I will blog on Chloroform Shock its relevance to my project.

Chloroform Shock is a method which is commonly used in the extraction of periplasmic proteins of gram-negative bacteria. Gram-negative bacteria is not only enclosed by its cell membrane, but also a periplasm. Many periplasmic proteins can be found within the periplasmic space (the region between the inner and outer membrane) of the bacteria. Hence, to extract these periplasmic proteins, chloroform is used.

Chloroform is a solvent which is relatively unreactive, miscible and volatile. It has to be said the exact mechanism of action of chloroform shock is currently not known. However, scientiests have been using this technique to extract periplasmic proteins. It is postulated that chloroform form pores in the outer cell wall of the gram-negative bacteria and releasing periplasmic proteins out into the extracellular environment. The optimal time for chloroform to work is 15 minutes for extract of periplasmic proteins only. If the time taken is too short, it may not have reached/penetrate the periplasmic space and periplasmic proteins are not extracted. When the time taken is too long, it may penetrate the periplasmic space and will extract other intracellular proteins as well, such as cell membrane proteins which is irrelevant to my project. Thus, great care must be taken to ensure reaction of the bacteria (cell pellet) with chloroform is 15 minutes exactly.

The use of chloroform is not without its risks. The bottle of chloroform is always opened within the Biosafety Level II Cabinet (which i am using) and not outside it. This is because accidental inhalation can be bad for the health. The role of the Biosafety Cabinet level II is to protect the user as well as the speciman. In Class II cabinets, there is always stream of inward air moving into the cabinet. This is called the inflow and prevents the aerosol generated during any microbial work, to escape out of the Cabinet (air can only flow into the BSC but not out). The inflow can only reach the front inlet grill, just in front of the operator. This is to ensure that unfiltered air outside the BSC cannot enter the BSC and thus there will not be any contamination. A special feature of BSC Level II cabinet is the HEPA-filtered air stream which causes air stream to flow downwards inside the BSC after sucking air from above and filtered. This flushing is called downflow and protects samples within BSC from contamination. In the case where chloroform is accidentally inhaled or consumed, it can depress the nervous system and cause dizziness, fatigue and headache. Hence the appropriate personal protective equipment to wear is a pair of gloves, labcoat and work inside the Biosafety Level II cabinet when performing chloroform shock.

Basic Outline of Chloroform Shock (sorry i cannot give the full details such as centrifuge speed, volume, etc as my supervisor do not allow, hence here is only the basic workflow)

1. Centrifuge to obtain cell pellet (from broth culture)
2. Wash cell pellet
3. Resuspend with PBS (phosphate buffer saline)
4. Repeat step 1 and 2 three times
5. Take OD600 reading
6. Calculate volume of cells to dispense into 5 eppendorf tubes
7. Centrifuge the 5 eppendorf tube
8. Decant supernatant and mix gently
9. Add chloroform and incubate 15 minutes
10. Add Tris-HCl
11. Centrifuge the eppendorf tubes
12. Extract the periplasmic proteins from the supernatant

Thanks for taking your time to read my entry and have an enjoyable and fruitful SIP for the next 12 weeks.

From: Ma Xianwei Benjamin
Class: TG01
0606181F

This would be my second posting about my SIP. Today I will be talking about how do you detect presence of Dengue antibodies in patient’s serum.

Firstly I will discuss about the introduction of Dengue virus. Dengue is a flavivirus, it is found in large areas of the tropics and subtropics. Transmission is by mosquito, principally Aedes aesgypti and Aedes albopictus. Dengue virus infection causes a spectrum of clinical manifestations ranging from symptomatic to fatal hemorrhagic disease. Classic dengue is characterized by sudden onset of fever, intense headache, myalgia, arthralgia and rash. A dysphasic febrile course is common, as it is insomnia and anorexia with bitter or loss of taste. Dengue hemorrhagic fever and dengue shock syndrome are severe complications often associated with secondary dengue infection.

In endemic regions, patients diagnosed with dengue fever generally have secondary infection. Consequently, detection of antibodies to dengue is valuable procedure, particularly in second and subsequent infections where the occurrence of complications is high. Traditionally, haemagglutination-inhibition (HAI) titers have been used to classify infections as primary or secondary. The current definition depends on an assay of paired serum specimens separated in time by at least 7 days, although any acute specimen with an HAI titer ≥ 1:1280 is defined as coming from a patient with seconday flavirus infections.

My company uses this instrument called Panbio Dengue Duo Cassette. It is for the qualitative presumptive detection of IgM and IgG antibodies to dengue virus in human serum. The assay can be used for the presumptive differentiation between primary and secondary infection. This test should only be used for patients with signs and symptoms that are consistent with dengue virus infection. Positive results are presumptive and must be confirmed by virus isolation, paired serum analysis, antigen detection by immunohistochemistry or viral nucleic acid detection for conifrmation of dengue virus infection.

The sensitivity of this assay has been set so that in patients with primary dengue, IgM is positive while IgG is negative. In contrast, patients with secondary infections will have a postitive IgG result with or without postive IgM result.

The principle of this assay, is when present in the patient sample, dengue-specific IgM or IgG antibodies bind to anti-human IgM or IgG antibodies immobilizied in 2 lines across the cassette membrane. Colloidal gold complexes containing recombinant dengue 1-4 antigens are captured by the bound patients IgM or IgG to give visible pink line(s). a procedural control is included to indicate that the assay has been performed correcty.

By Ivan Ng
TG010605070B
12.15pm
4 August 2008 (Mon)

Week 6 SIP

Hi,

Week 6 of SIP. I will share my SIP experience chronologically. I am still at histopathology department.

Week 2
There was a talk on how to handle spillage. It was briefed to all staff. Training staff on how to handle chemical spills is important because some chemicals are potentially harzardous or flammable. The key point to remember from the talk is MSDS. Material Safety Data Sheet. It contains all the necessary information regarding the chemical.

Next there was a briefing during emergencies, particularly an outbreak of fire. We were taught on fire safety procedures and evacuation route. After these 2 briefings, there was a small quiz for all staff, which was required by JCI. I scored 12/14.

Week 2 was about the same as the first week. I heated slides to melt the wax, labelled casettes and sorted slides. We also had a rough idea on our major project. To find out the minimum processing time.

Week 3
I labelled chemical bottles in the cabinet. We have to check the expirary date, date of opening and batch number. I was assigned to do this because there is a JCI inspection next week. We have to ensure all chemicals and materials are accounted for and not expired.

Apart from checking chemicals, I did slide sorting for the whole week. A recap: we have to sort according the the biopsy number eg. 08-12345, then followed by the block number eg. A3, then by the number of levels being cut. eg. VL 2.

For example, this is how we arrange it.
(A1 vl 1/3 08-19000)
(A1 vl 2/3 08-19000)
(A1 vl 3/3 08-19000)
(A2 08-19000)
(B1 08-19000)
(A1 08-19001)
(A2 vl 1/2 08-19001)
(A2 vl 2/2 08-19001)
(A1 08-19005)

Notice that some blocks do not have "vl" because the block is only cut once. Meaning only one layer is cut. vl stands for variable level. The 08 in the biopsy number 08-xxxxx, stands for the year, 2008.

Apart from sorting, I rearranged boxes of blocks in the store room. We have to keep blocks for at least 5 years. Just in case the doctors requires a re-examination when a patient suffers a relapse or the doctor made a mistake 5 years ago.

Week 4
JCI inspection week. We didn't do much work this week because the auditors from JCI may come any day. JCI inspection occurs once every 2 years. I heard from one of the staff that there are 3 standards in JCI acceditation, a basic level, silver and gold. Currently, my lab is at the silver standard. After this inspection, it may become gold.

We also managed to collect some tissues this week for our project. But unfortunately for JCI, we weren't able to use it. Furthermore, our machine temporarily malfunctioned on friday.

Week 5
Besides heating and sorting slides, I have started shaving blocks. There are about 500-700 blocks in a day and it is rather tiring to shave all of them. It is fun because I can finally get to use the microtome. Oh and I accidentally cut my finger, fortunately only the skin peeled off, no bleeding. The blade is very very sharp.

Shaving is done at 20 um instead of the usual 4 um that we use for sectioning.

I was on MC one of the days. Apparently, there is a flu bug going around the lab where more than 5 staff have gotten sick, including ying chee. And on Friday, we went back to school for campus discussion. So this week I wasn't around much.

Week 6
Shaving of blocks, fishing, sorting slides, heating. We have started processing our tissues that we collected for our project. it was rather sucessful. One of the staff briefly verified our slides and commented that it was acceptable. We have successfully shortened the time!

I have growned to like shaving very much. Eventhough it requires speed and muscle, my job is very important and we have to be alert, so as not to cut too much or too little.

I have also observed how embedding is performed. It is quite easy for standard tissues but for postate and equally tiny tissues, it is rather difficult. Imagine a short hair. We have to use a forcep to carefully pick it up and put it in molten wax. In addition, we have to press it down using a screw-driver like instument. The trouble is, every time after we press it down, we have to remove the instrument, and the tissue always move away.

Thank you
Ernest
TG01
02/08/08

Week 5 SIP/MP Experience

Hello all, its been 5 since SIP/MP started and it was great seeing you all on friday again. This week it is my turn to share my SIP/MP experience to you all.

I am attached to a plant biotechnology lab, and under the TSO, I help in making plant media. I make two kinds of plant media. One has activated charcoal in it and one does not. The steps to making the plant media are similar, therefore I shall explain one, and list the constituents of the other.

Preperation of tissue plant media
1.) Fill up two 3Litre beakers to over 2.5L with MilliQ water.*
2.) Place the beakers on seperate magnetic stirrers.
3.) Add the constituents in the following order after weighing them/determining their volume in each beaker. (MS media formulation (Murashige and Skoog)-6.45g; Myo-30ml; Organic elements-30ml sucrose 60g).*
4.) Top up the water until both beakers are 3L full.
5.) Next the pH is adjusted to a range of 5.2 to 5.21 using the pH meter
6.) Activated charcoal(1.5g) is added next and allowed to be distributed in the media.**
7.) Next 30 vitro vents(containers that will carry the media) are prepared.
8.) 1.6grams of agar powder are then poured into each vitro vent.
9.) Next a marked measuring cylinder specially used to measure for the volume needed for each vitro vent is used and the media is poured to fill up all the 30 vitro vents.
10.) The vitro vents are then covered with a cover and autoclaved.

Constituents for second set of plant media
1.) MS 6.45g
2.) Myo 30ml
3.) Organic Elements
4.) Sucrose 30g
5.) pH 5.2-5.21
30 vitro vents
1.6g agar

*Avoid parallax error by placing the eye onthe same level as the meniscus and read the bottom of the meniscus
**The activated charcoal does not dissolve into the media, therefore it must be thoroughly in the media before it is poured into the vitro vents. And after half the beaker is emptied, place the beaker back on the magnetic stirrer to redistrubute all the activated charcoal that has settled down.

I hope my explanation is clear and easy to understand. If there are any questions, please feel free to ask me. Thanks for taking the time to go through this post.


Johan
0606637G
TG01

Week 4 SIP/MP Experience

Hello my fellow coursemates! How are you guys doing? Hope all of you are doing fine for your SIP and MP. This is the 4th week of SIP/MP and it is my pleasure to share my attachment experience.

I am attached to a research laboratory which study on the effect of polyphenolic chemosensitisers and anticancer drugs on tumour cells. Some examples of anticancer drugs include melphalan, chlorambucil and etc. Both chemotherapy drugs belong to the same class of nitrogen mustard alkylating agents. Melphalan is commonly used to treat multiple myeloma (cancer of plasma cells) and ovarian carcinoma. Chlorambucil is used primarily to treat chronic lymphocytic leukemia (cancer of lymphocytes, with particular to B cells). Some examples of polyphenolic chemosensitisers include 2,2'-dihydroxychalcone and 2'-hydroxy-4-methylchalcone (I shall elaborate more on polyphenolic chemosensitisers in my next post).

In the world today, there are 3 main types of cancer therapies which are chemotherapy, radiotherapy and surgery. Usually, they are used in combination to achieve greater effectiveness in treating tumours. My main focus will be on chemotherapy.

Chemotherapy is decreasing in its effectiveness mainly due to chemoresistance of tumour cells. This is primarily due to the gluthatione (GSH)-related detoxification system which allows tumour cells to be resistant to chemotherapy drugs. I shall elaborate further on this system in my next post.

In my study, I shall be focusing on the effect of 2,2'-dihydroxychalcone and chlorambucil in inducing DNA interstrand cross-links on human colon adenocarcinoma cells (HCACs) grown in-vitro. The HCACs are obtained from ATCC (American Type Culture Collection) and designated by COLO 320 HSR. These cells are grown in RPMI (Roswell Park Memorial Institute) medium and they are loosely adherent to the culture surface. In addition, they appear round and refractile under the inverted microscope. Since they are tumour cells, they divide very rapidly, and thus have high energy requirement.

During the course of my research, I am required to maintain these cells in peak condition so that I will have enough cells to continue my experiments. Hence, I will be applying what I have learnt from Mammalian Cell Technology (MCT) which I have taken as elective subject to part of my research. Without further delay, let me start off with the most fundamental, which is the preparation of the RPMI-1640 medium.

Subject: MCT
Tests:
1)Preparation of RPMI-1640 medium
2)Subculturing of cells
3)Changing of medium


1) Preparation of RPMI-1640 medium

Composition

· 1% sodium pyruvate
· 1% non-essential amino acids
· 1% antibiotics
· 10% fetal bovine serum (FBS)

Materials

· 500ml RPMI-1640 medium (ready-to-use)*
· Fetal Bovine Serum (FBS)*
· Antibiotics*
· Sodium pyruvate*
· Non-essential amino acids (NeAA)*
· BSC 2
· 70% ethanol
· Paper towels
· Pipettorˆ
· Falcon™ pipette tubes (5ml)ˆ

Methods

A. Preparation of BSC 2

1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.

B. Preparation of RPMI-1640 medium

1. Place the materials* in 37ºC waterbath.
2. Place the materials* into the BSC 2.
3. Pipette 5ml of sodium pyruvate into the pre-made 500ml RPMI-1640 medium.
4. Pipette 5ml of NeAA into the pre-made 500ml RPMI-1640 medium.
5. Pipette 5ml of antibiotics into the pre-made 500ml RPMI-1640 medium.
6. Pipette 50ml of FBS into the pre-made 500ml RPMI-1640 medium.
7. Store the medium at 2-8 ºC and warm up in 37ºC waterbath before any usage.

Function of components:

· Sodium pyruvate- Provide additional source of energy for rapidly-growing cells.
· NeAA- Provides source of amino acid (energy source for cells).
· Antibiotics- Minimises bacterial contamination of tissue culture.
· FBS- Provides essential nutrients to promote optimal cell growth and proliferation

2) Subculturing of cells

Principle

As cells grow and multiply, they tend to crowd together, which is termed as confluency. This can be observed under the inverted microscope where cells are in close proximity. A confluent tissue culture flask suggests the need for subculturing. Subculturing is defined as the inoculation of cells from a confluent flask into a new sterile flask with fresh medium. It allows cells to have more growth surface so as to minimize the competition for growth surface and nutrients. Subculturing is especially important for maintaining the viability, growth and proliferation of anchorage-dependent cells as these cells need to adhere to the surface of the tissue culture flask before they can start growing.

Materials

· 75cm² tissue culture flask (containing HCACs of 80% confluency)
· New 75cm² tissue culture flaskˆ
· RPMI-1640 medium*
· 0.0067M phosphate buffered saline (PBS)*
· Trypsin*
· Inverted light microscope
· BSC 2
· CO2 incubator
· Pipettorˆ
· Sterile 50ml Falcon™ tubeˆ
· Falcon™ pipette tubes (25ml and 5ml)ˆ
· Waste beakerˆ
· Biohazard bag
· Clorox (Bleach)
· 70% ethanol
· Paper towels

Methods

A. Preparation of BSC 2

1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.

B. Preparation of reagents

1. Incubate trypsin and RPMI-1640 medium in 37ºC water bath.

C. Washing of HCACs

1. Remove the 75cm² tissue culture flask (containing HCACs of 80% confluency) from the CO2 incubator.
2. Observe cell confluency (80%) under inverted light microscope.
3. Swab the 75cm² tissue culture flask with 70% ethanol before placing it into the BSC 2.
4. Swab the materials* before placing them into the BSC 2.
5. Discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker.
6. Pipette 10ml of 0.0067M PBS into the 75cm² tissue culture flask to wash the HCACs.
7. Swirl the 75cm² tissue culture flask gently to facilitate the washing of HCACs.
8. Discard 10ml of PBS into the waste beaker.

D. Trypsinisation

9. Pipette 2ml of trypsin into the 75cm² tissue culture flask to detach the HCACs from the tissue culture flask surface.
10. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2 for 2-3 minutes.
11. Observe the 75cm² tissue culture flask under the inverted light microscope to ensure the HCACs are detached.

E. Subculturing of cells

12. Pipette 8ml of fresh RPMI-1640 medium into the 75cm² tissue culture flask to neutralise trypsin to prevent damage to the HCACs.
13. Mix the RPMI-1640 medium with trypsin well to ensure all the HCACs can be transferred with the RPMI-1640 medium into a sterile 50ml Falcon tube.
14. Pipette 10ml of mixture (8ml of RPMI-1640 medium and 2ml of trypsin) from the 75cm² tissue culture flask into a sterile 50ml Falcon tube.
15. Centrifuge the sterile 50ml Falcon tube at 20ºC, 1500rpm for 3 minutes.
16. Discard the supernatant into the waste beaker, leaving the cell pellet.
17. Resuspend cell pellet in 10ml of PBS.
18. Centrifuge the sterile 50ml Falcon tube at 20ºC, 1500rpm for 3 minutes.
19. Discard the supernatant into the waste beaker, leaving the cell pellet.
20. Resuspend cell pellet in 10ml of RPMI-1640 medium.
21. Pipette 19ml of fresh RPMI-1640 medium into a new 75cm² tissue culture flask.
22. Pipette 1ml of cell suspension from the sterile 50ml Falcon tube into a new 75cm² tissue culture flask.
23. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2.

F. End of experiment

1. Deactivate the waste solution in the waste beaker with Clorox.
2. Drain the waste solution into the sink after it turns from violet to colourless and run running tap water for 5-15 minutes.
3. Dispose all the used Falcon™ pipette tubes and paper towels and into the biohazard bag.
4. Swab the BSC 2 with 70% ethanol.
5. Return all the reagents to their appropriate storage areas.

3) Changing of medium

Principle

As cells grow and multiply in culture medium, they produce metabolic toxic waste products and use up nutrients in the medium. Essentially, we want to maintain the viability of cells and thus, the changing of medium is necessary as it restores back the level of nutrients and removes metabolic toxic waste products. An indication of the need to change medium is by observing the colour of the medium. Phenol red is added in the medium to act as a pH indicator. A yellow culture medium indicates the accumulation of metabolic toxic waste products and depletion of nutrients, prompting the need to change medium. A cloudy culture medium suggests possible bacterial contamination and the best action would be to discard the tissue culture flask.

Materials

· 75cm² tissue culture flask (containing yellow culture medium and cells)
· 0.0067M phosphate buffered saline (PBS)*
· RPMI-1640 medium*
· BSC 2
· CO2 incubator
· Pipettorˆ
· Falcon™ pipette tubes (10ml and 25ml)ˆ
· Waste beakerˆ
· Biohazard bag
· Clorox (Bleach)
· 70% ethanol
· Paper towels

Methods

A. Preparation of BSC 2

1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.

B. Changing of medium

1. Remove the 75cm² tissue culture flask (containing HCACs of 80% confluency) from the CO2 incubator.
2. Swab the 75cm² tissue culture flask with 70% ethanol before placing it into the BSC 2.
3. Swab the materials* before placing them into the BSC 2.
4. Discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker.
5. Pipette 10ml of 0.0067M PBS into the 75cm² tissue culture flask to wash cells.
6. Swirl the 75cm² tissue culture flask gently to facilitate the washing of cells.
7. Discard 10ml of PBS into the waste beaker.
8. Pipette 20ml of fresh RPMI-1640 medium into the 75cm² tissue culture flask.
9. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2 for 2-3 minutes.

C. End of experiment

1. Deactivate the waste solution in the waste beaker with Clorox.
2. Drain the waste solution after it turns from violet to colourless.
3. Dispose all the used Falcon™ pipette tubes and paper towels and into the biohazard bag.
4. Swab the BSC 2 with 70% ethanol.
5. Return all the reagents to their appropriate storage areas.

In conclusion, subculturing of cells and replacement of medium is essential for maintaining the viability of cells. Alright, that is all for now! I hope my post is comprehensive and if there is any query, please feel free to ask.

Thankz! =)

Tan Han Yang
0606190G
TG01