Lab Techniques

Hi everybody! I'm attached to a research center which all of you are VERY familar with. =D My MP and SIP are done together with Ming Boon, Johanna & Shahirah. Our research revolves around proteomic (protein-related studies) work and the bacteria that we are looking at is Stenotrophomonas maltophilia. It is a Gram-negative bacillus which causes nosocomial infections. We are working on 2 different strains, the environmental (from soil) and clinical (from patient infected with S.maltophilia). Hopefully, we will be able to identify the difference in protein composition between the two strains, which makes the clinical strain infectious.

As you know, a cell is made up of many different kind of proteins; secretory, cell membrane, cytoplasmic and etc. So, it would probably take years to study and analyze all of them! We were given a visable goal which is to look into only 2 types of protein; secretory and exposed cell membrane proteins. These 2 types of proteins are selected as a microbe usually attaches to and infects a cell through secreting proteins or sticking onto it through the exposed cell proteins. At this point of time, we are still analyzing the secretory proteins.

The secretory proteins alone consist of many different proteins. In order to analyze individual proteins, they first have to be separated. For our research we're using the 2-Dimensional Electrophoresis approach.


First Dimension Separation : Isoelectric Focusing (IEF)

Proteins are amphoteric molecules such that they can carry either positive, negative or zero net charge depending on the pH of their local environment. For every protein, there is a specific pH at which its net charge turns zero. This is its pI. pI refers to isoelectric point and is defined as the pH at which a protein will not migrate in an electric field and is determined by the charges it carries.

The proteins are placed in a medium with a pH gradient. When electric field is applied, they will move towards the electrode with opposite charge (unlike pole attracts). During migration through the pH gradient, proteins will either gain or lose protons leading to decrease in net charge and mobility. Ultimately, the protein will reach a point where the pH gradient is equal to it's pI. Being uncharged, migration would stop. In any case where a protein were to diffuse to a pH region lower than its pI, it will be protonated and forced back towards the cathode by the electric field. On the contrary, if it diffuses to a region of pH gradient greater than its pI, the protein will be negatively charged and move towards the anode. Through this way, focused spots are obtained.

P.S. The circles represents the proteins and the number
within it is its pI

Adapted from: http://www.bmskorea.co.kr/bms_product/bms_Product_Sec/bms_Product_Sec_List.aspx?sec=sec&cstep=3&cgroup=32

For our project, the pH gradient used is a strip of acrylamide matrix gel that has pH gradient incorporated covalantly making it immobolized even under electric field. These strips will have to be rehydrated with rehydration buffer and protein samples, together with the buffer, the proteins to be separated will be absorb into the gel strip. The gel strip used is called IPG strip.


Second Dimension Separation : SDS PAGE (Molecular Weight)

The pH gradient strip is then placed in the well of the acrylamide gel for 2nd dimension separation.

The proteins on the pH gradient gel migrates down, separation based on molecular weight.

Reminder: The numbers refers to protein's pI

The black spots indicates the stained and separated proteins

Adapted from: http://www.biosciencetechnology.com/ShowPR.aspx?PUBCODE=090&ACCT=9000012495&ISSUE=0401&RELTYPE=PR&PRODCODE=00002750&PRODLETT=B


The protein spots will be excised with Xcise (scroll down to view Ming Boon's post) and analyzed with MALDI (scroll down to view Shahirah's post).

THAT'S ALL for my post! =D


Tang Jiaxin
0503257H
TG01

Lab Technique

Hiya, I’m currently attached to clinical pharmacology lab doing research work. Most of my work involves PCR techniques and DNA sequencing. I’m currently doing my SIP and MP with Zahirah. Today, I’m going to share with you guys an experiment, which is important because it will affect our subsequent experiment results.

Experiment title: Finding the optimal temperature of primers.

PCR-Materials and Method

-It is good to cultivate a good practice to prepare a mastermix.

Reagents Required:	Volume Required/ul:	Mastermix Volume/ul:
Autoclaved water	6.4	89.6
Buffer	1.0	14.0
MgCl2	0.6	8.4
dNTP	0.3	4.2
Forward Primer	0.2	2.8
Reverse Primer	0.2	2.8
Test DNA Sample	1.0	14.0
Taq Polymerase	0.3	4.2
	10.0	140

-Add all the required reagents into a large 1.5ml eppendof tube(This is your mastermix.), and pipette 10.0ul into each 0.2ml PCR tube. These reagents must be placed in a ice-box or chiller to prevent degradation of the components in the reagents, particularly Taq Polymerase. Taq Polymerase is to taken out only when it is required and it must be returned back into the fridge immediately. Ensure the mastermix is well-mixed by centrifuging and vortexing it for a few seconds.

-Place them into the PCR machine in one straight row because different columns have different temperature. We are performing gradient PCR in which we do not know the annealing temperature for the primer hence we have to program the annealing temperature range to be from 50C to 60C and the machine will set up a specific temperature for each different column. The PCR conditions are initial denaturation at 95C for 3 minutes, denaturation at 94C for 45 seconds, annealing at the temperature range being set for 45 seconds, extension at 72C for 45seconds and final extension at 72C for 7 minutes. The PCR conditions are repeated for 30-35 cycles to have sufficient amount of particular segment of the gene that is required.

-After the PCR process is done, we must perform gel electrophoresis and operating the Bio-imager to view the DNA image. After analyzing the DNA image (Thickest band.), we must check the specific temperature of the column which the thickest band is seen from. This temperature will be the optimal temperature of the primer.

Refer to Zahirah’s posting regarding on gel electrophoresis as she will explain it in details. Hope you have learnt something from my SIP and feel free to ask any questions ya.

Posted by: Michelle TG02

Lab Techniques

Topic: MALDI TOF (matrix-assisted laser desorption/ionization-time of flight)

As mentioned earlier in Ming Boon's post, the MALDI TOF is used in the analysis of the proteins that were initially separated through 2D gel electrophoresis and excised using Xcise. The peptides are then spotted, together with the matrix compound, on a 384-well MALDI plate (sample plate). Unlike Xcise, there was no formal training for us on the use of the 4800 Plus MALDI TOF/TOF Analyzer.

In this post, i will try my best to explain more on the principles of MALDI TOF.

The samples are analyzed in a two-step process. During the first analysis, the samples (peptides) are ionized using laser. When the laser hits the spot (contains the peptides mixed with the matrix) on the sample plate, it will cause the matrix to be ionized. The ionized matrix will transfer its ion to the sample. This ionizes the peptides. Another beam of laser is flashed onto the spot with a high voltage applied to the sample plate. This will cause the ionized peptides to accelerate out of the plate and into the flight tube.

The ions are accelerated with the same potential (energy) and form up at a fixed initial point and time. (It is like a race where all the runners form up at the starting line.) The ions are then allowed to drift down the flight tube. They will separate base on their mass to charge ratio, the lighter ions will have a higher velocity while the heavier ions will have a lower velocity. The time taken for an ion to travel from the start to the end, where the detector is, through the flight tube is measured i.e the time of flight (TOF). The m/z value is then obtained. That is for the first part of the analysis.

The second part of the analysis will involve the fragmentation of ions analyzed earlier. The form of fragmentation used in this analyzer is known as Collision Induced Dissociation (CID). The ions (parent ions) are selected to enter the collision cell based on the parameters set earlier. Inert gas molecules are introduced into the collision cell at a certain pressure. The ions are then selected by the precursor ion selector and transmitted into the cell. The gas molecules and ions will collide with one another and during the collision, there will be transfer of energy which causes the fragmentation of the parent ion. This will give rise to daughter ions which are then subjected to analysis. Like the parent ion, the daughter ions will be analyzed by measuring the time of flight and the m/z value is then obtained. [This also explains for the double TOF seen in the name of the analyzer.]

During the analysis, the measurements (m/z values) are shown as peaks. At various peaks, it is checked if it meets certain criteria like resolution, which will impact the following steps. When a parent ion is fragmented, it gives rise to daughter ions.These ions can be detected from the peaks. They can be b-ions (charge remains on the N-terminal of the peptide) or y-ions (charge remains on the C-terminal of the peptide). B-ions comprise of an amino acid (eg lysine) and a proton (hydrogen ion) while y-ions are made up of an amino acid and water. The amino acid sequence is deduced by the difference in mass between consecutive ions which corresponds to the mass of the individual amino acid.

That will be all for my post. I apologise if my post is unclear or kinda choppy in terms of the content. I will try my best to answer any queries that you guys may have aite? Till then enjoy your SIP & see all of you on Friday! Cheers!

Posted by Shahirah Bibi, TG01, 0503174E.

Microbiology/LMQA

Topic: Blood Culture

Posted by: Azhar Hamdan TG01 (0503269C)

Equipments & Materials:

• BACTEC™ Fluorescent series blood culture system
• BACTEC™ Plus Aerobic/F Culture Vials Soybean-Casein Digest Broth (Blue Cap)
• BACTEC™ Plus Anaerobic/F Culture Vials Soybean-Casein Digest Broth (Gold Cap)
• Aerobic venting unit
• Class II Biosafety Cabinet
• Calibrated inoculating loops
• Blood Agar Plate (BAP)
• MacConkey Agar (MAC)
• CDC Anaerobic Blood Agar (CDC)
• Sterile 70% ethanol pads (i.e. Alcohol Swab)
• Glass slides
• Gram stain (Crystal violet, Gram’s Iodine[Mordant], Acetone[Decolouriser], Safranin)
• Tubes of pre-prepared 1mL saline
• Tubes of pre-prepared 200µL rabbit plasma

For this posting, I am focusing more on the sub-culturing of blood onto plates. For more information on the BACTEC machine and how it works, refer to BMTjournal (Boon Ching’s Posting on Blood Culture). Linked with permission from Boon Ching.

Basically, the flow would be, BACTEC™ machine THEN sub-culturing of positive vials.

Procedures

Detection:

When the BACTEC machine detects a positive sample, the positive vial (which contains blood and can either be under Aerobic or Anaerobic conditions) is taken out. This is done through the scanning of a barcode at the door of the machine to indicate to the machine that you are removing a vial, followed by the scanning of the vial’s barcode.

The vials have 2 barcodes; one barcode is the original pre-generated BACTEC™ barcode while the other barcode is a pre-generated barcode that is provided by the lab. The laboratory barcodes comes in sets of 2. One of the pair is pasted onto the bottle and the other is pasted onto the request forms.

Following the removal of the vial, the request form is singled out, and qualified staff with access to the Laboratory Information System (LIS) will flag the vial as positive.

BACTEC machine interior

Barcodes located at the door of the machine. These are scanned to tell the machine whether you are adding vials or whether you are removing positive or negative vials

Sub-culturing:

After flagging, 4 labels identical to the vial’s lab barcode numbers are generated. Each of the labels is pasted onto BAP, MAC, glass slide and 1mL saline tube. For an Anaerobic vial, an additional label is generated, with an ‘A’ after the number and this is to be pasted onto a CDC plate. (e.g. DB43210 for the 4 labels, and DB43210A for the Anaerobic plate)

Before anything is done, the cover of the vial and the glass slide is wiped with alcohol swabs. The glass slide is marked with a circle to indicate later on, the area of the drop of blood.

All of the materials, including the vial, are placed in a Biosafety Cabinet (BSC), and the whole process of sub-culturing is done within the BSC. This is to prevent the accidental exposure to aerosols from the blood.

A venting unit is attached to the vial, which would allow the blood to be slowly dripped out for testing. Firstly, 1 drop of blood is dripped each on BAP and MAC. For an Anaerobic vial, an additional drop of blood is dripped onto a CDC plate. Next, an inoculum with 6-fold dilution is obtained by adding 8 drops of blood to the tube of 1mL saline (8drops is roughly 0.2mL). Lastly, 1 drop of blood is dropped onto a glass slide and the drop is spread with an inoculating loop within the pre-indicated circle. Sometime 2 different specimens can be dropped on the same slide to avoid wastage.

The BAP, MAC and CDC (if used) are to be streaked later on after the drop of blood has partially dried. Furthermore, the BAP is streaked with Staphylococcus aureus. BAP and MAC are incubated at 35° in CO₂ for 24 hours whereas the CDC is incubated under anaerobic conditions at 35° for 48 hours before reading. The inoculum (saline with 8 drops of blood) is passed to the investigation lab to be further tested. The blood on the glass slide is allowed to dry on a hotplate within the BSC, before it is gram stained.

Method for streaking
(Also shows the S. aureus streaks done on BAP plates)

BAP that have been incubated at 35° in CO2 for 24 hours

Slides for Gram Stain (Left: Single slide, Right 2 specimens on one slide)

Gram Staining

• Stain with Crystal Violet for 1min
• Flush with water
• Counter-stain with Gram’s Iodine for 1 min
• Flush with water
• Decolourise with Acetone for 2 sec
• Flush with water
• Stain with Safranin for 1 min
• Blot dry

After staining, a presumptive preliminary identification of the organism is done on the basis of the organism being either gram positive or negative or whether it is either a bacilli or cocci. This preliminary identification is crucial for doctors as it allows them to start the patient on the relevant antibiotics.

Direct Tube Coagulase Test

This is done for Gram Positive Cocci (GPC) as a confirmatory test to check whether the GPC is S. aureus. 60 µL of blood (about 3 drops) is added to 200µL rabbit plasma and incubated for 4 hours to overnight.

As S. Aureus is capable of coagulating rabbit plasma, a positive coagulase test would suggest that the GPC is S.Aureus. Further tests are done in the investigation lab, to confirm the identification of S. Aureus.

Thats all for this posting, feel free to ask me questions. Images posted here were taken with permission from laboratory supervisor. He actually encourages us to take photos because he believes that visuals are more important in learning therefore naturally me and the others have been taking pictures non-stop, a fair bit of which is totally unrelated to learning, haha. Anyways, lets all enjoy the rest of our remaining 17 weeks of SIP :) .

Lab Techniques/LMQA

Name of topic: Xcise

Content of Topic:

My MP involves the analysis of Stenotrophomonas maltophilia’s secretory and cell surface proteins. This would involve the use of quite a number of machines. The one that I am going to blog about is Xcise. We were given a training that lasted for 3 days. The first day is a trial run, the second day is about how to use the software and the last day is more on the maintenance of the machine.

Xcise is an automated gel processor that is able to process proteins that are to be identified by mass spectrometry, from the acquisition of gel image to the spotting of protein sample onto a MALDI target plate.

This picture is taken from http://www.ssi.shimadzu.com/products/images/Biotech/xcise.jpg

Terms:

2D gel electrophoresis: Proteins are separated twice. For the 1^st dimension, proteins are separated using an IPG (Immobilized pH Gradient) strip based on their isoelectric point. The proteins move horizontally. For the 2^nd dimension, proteins are separated using a pre-casted gel based on their molecular weight. The proteins move vertically downwards.

Isoelectric point: Isoelectric point is a characteristic of the protein whereby it corresponds to the pH at which the protein is neutrally charged.

Mass Spectrometry: A technique used to identify and sequence proteins by measuring the mass-to-charge ratio of proteins that are converted to ions. The instrument used to measure the mass spectrum is called MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight).

Matrix compound: A compound that is required to control the energetics of the desorption/ionization process.

After a gel is run, proteins are separated based on their isoelectric point and molecular weight (2D gel). Proteins separated would appear as spots on the gel. The protein spots would then be stained to be visualized and gel image is acquired. After the gel is stained, we would want to identify the proteins. Before running the proteins through MALDI, the proteins would need to be removed from the gel, digested into peptides and then spotted onto a MALDI target plate.

Outline of Xcise’s in-gel digestion procedure

1. Gel image is acquired and spots to be cut are selected
2. A cutting head will cut out the gel containing the protein spots and place them into wells
3. The gel will then be destained and dehydrated
4. Trypsin is added to digest the proteins into peptides (note that the proteins are still within the gel)
5. Sample is then incubated for 4-6 hrs at 37^oC or 16-18 hrs at 30^oC
6. As buffer used contain salts that will affect the subsequent analysis of peptides in MALDI, peptides would need to be desalted
7. Peptides are then eluted using ZipTip. ZipTip is different from a normal pipette tip as it contains a resin at the tip. Peptides would bind to the resin during the process of desalting
8. The peptides are then spotted onto a MALDI plate together with a matrix compound
9. The MALDI plate can then be placed in MALDI and peptides can be analysed

LMQA – Lab Automation

Using of Xcise is an example of lab automation. If the process is to be done manually, it will be very tedious. For the cutting of gel, the lab technician would need to measure the diameter of the protein spot, cut a pipette tip so that the hole corresponds to the diameter and use the pipette tip to cut the gel. The gel would be inside the tip and would have to be taken out using a pointed end. Steps 3-7 would need to be done manually too by adding and removing the required solutions. For the spotting onto MALDI plate, lab technician would have to spot onto the plate one by one. One spot will be placed in one eppendorf tube so if there are 100 spots, there will be 100 tubes to be processed and 100 spots to be spotted onto the plate.

However, if Xcise is used, the cutting of gel would be faster and more precise. At the spotting stage, Xcise can spot 8 samples at a time, which is much faster and accurate than doing it manually. Contamination is also greatly minimized. So even though the machine and the consumables are very expensive (1 ZipTip costs about $2, and 8 ZipTips will be used at a time), it can greatly improve the efficiency of the lab.

This picture is taken from http://www.millipore.com/micro/jp/zt/ziptip.gif

That is all about it. Feel free to ask any questions. Thanks=]

Chua Ming Boon
Tg01
0503197F

Cytogenetics (Lab technique)

Name of test: in situ culture technique for chorionic villi samples

Principle of Test: The tissue is desegregated into a single cell single cell suspension by means of enzyme or mechanical disruption. These single cells are plated onto a sterile glass coverslip in a petri dish where they grow in situ to form colonies.

Clinical interpretation: Test for foetal abnormality.

When sample comes in, the first thing to be done is requisition. These are the main information stated in the request form:

Samples Collected: Chorionic villi specimens

Clinical indication: Nuchal Translucency (NT) 6, 7mm. 1:2 (T21). 1:7 (T13:T18)

Possible Diagnosis: Foetal Abnormalities - Down’s syndrome (Trisomy 21), Edwards syndrome (Trisomy 18) and Patau syndrome (Trisomy 13).

Given this information, the cytogenetist have to culture, followed by harvest and banding, then lastly, analyse the chromosomes karyotype to confirm the diagnosis.

Before I continue, I would like to explain on certain terms.

Chorionic villi – a small sample of chorion or placental tissue for prenatal diagnosis, usually by aspiration biopsy with a thin plastic catheter inserted trans-cervically or trans-abdominally into the uterus. This tissue can be obtained in early pregnancy, thus allowing early detection of genetic abnormalities. This is usually performed at 10-12 weeks of pregnancy, whereas, amniocentesis (amniotic fluid) is performed after 14-15 weeks.

Nuchal Translucency – small transparent area beneath the skin at the back of the foetus’ neck. An increase in nuchal translucency measurement indicates an increase in the risk of chromosomal abnormalities, such as Down’s syndrome.

Trisomy – extra chromosome in the respective position eg, T13 means 3 copies of chromosome at chromosome 13 instead of usual 2 copies.

Now, I’m going to briefly explain the procedure of handling chorionic villi samples.

Culture

- Upon receiving the tissues, wash it with complete α-MEM / Amniomax™ - II medium (α-AM)

- Dissect under dissecting microscope to choose the appropriate fetal tissue

- Strip the outer layer to remove maternal tissues

- Clean the choronic villi tissues with 3ml of trypsin EDTA (10X concentration).

- Incubate it for 25 minutes, 37°C

- Centrifuge it at 1200rpm for 10 minutes

- Remove the supernatant (trypsin EDTA) and add in 1ml of collagenase into the tube with the pellet. Trypsin EDTA and collagenase aids in the digestion of various proteins to dissociate the tissues and obtain cells.

- Incubate it for 30-90 minutes, 37°C

- Single cells will be form and add in 3ml of α-AM (medium). This is to neutralise and stop the collagenase activity.

- Centrifuge it at 1200rpm for 10 minutes

- Prepare 4 culture dishes and label it as ‘A’, ‘B’, ‘C’ and ‘D’.

- Resuspend the pellet with 2ml of α-AM (medium) and transfer 0.5ml of cell suspension onto each coverslip in each of the 4 dishes.

- Culture ‘A’ and ‘C’ in one incubator while ‘B’ and ‘D’ in another incubator. This is one of CAP’s regulation and also to prevent failed cultures upon power failure.

The next day, flood culture ‘A’ and ‘B’ with α-AM while ‘C’ and ‘D’ with BioAMF2 (another medium). This is to optimise the growth as different cells may favour different medium.

Incubate the cultures for 5 days and then, check for attachment.

Harvest

Once more than 4 colonies or 50-200 cells can be seen, the culture can be prepared for harvest. During harvest, Colcemid® working solution is added to prevent the synthesis of spindle fibres and this arrest the cells at metaphase stage. Colcemid® is a cytotoxic chemical, therefore, it has to be handled properly and disposed into the designated cytotoxic waste.

Warm 0.8% sodium citrate hypotonic solution is added to increase the cell volume by swelling it, spreading the chromosomes apart. This enlarges the view of chromosomes.

1:3 methanol – glacial acetic acid fixative is needed to fix the cells, in order to preserve the architectural structure of the cells and prepare for staining. Giemsa & Wright’s stain is usually used.

Overall, It takes about 14 days to go through the process of Culturing -> Harvesting -> Banding -> Analysing.

Done by: Yvonne Lau

5 RED HOT diseases!

.....

okay okay! We've changed our narrowed down diseases through relating the patient's conditions and symptoms to the characteristics of the respective diseases. Subsequently, we are going to narrow down the diseases to 2! Sneak a peep at our 5 rash-eey RED and feverish-ly HOT diseases.

i Endemic Typhus

There are 2 causative agents for Endemic Typhus: Rickettsia typhi and Rickettsia felis. It is similar to epidemic typhus however it is milder in comparison.

1. The old man’s flat is infested with fleas and he has a habit of feeding stray cats and dogs thus transmission of Rickettsia typhi or Rickettsia felis from dog and cat fleas is highly possible.

2. Symptoms include high fever, headache and rashes.

ii. Leptospirosis

Leptospira interrogans is the causative agent of Leptospirosis.

1. The disease is widely found in Singapore

2. It is transmitted from animals to human through water, food and excreta of infected animals and since the old man has bad personal hygiene and a habit of feeding stray dogs and cats, he could have easily contracted the disease

3. Symptoms include fever, rash and vomiting.

iii. Lyme Disease

Borrelia burgdorferi is the causative agent of Lyme disease.

1. It is transmitted by bites from ticks, found on animals such as dogs, which eventually causes rashes and since the old man has a habit of feeding stray dogs, this is very possible.

2. Symptoms include fever, a characteristic rash and general weakness.

iv. Typhoid Fever

Salmonella typhi is the causative agent of Typhoid Fever. Typhoid Fever and Endemic Tyhpus is not related.

1. S. typhi is part of the normal flora in our body and thus could easily have been transmitted to the old man.

2. The old man’s mattress are soiled human excreta and urine thus he could easily get infected since infection occurs through accidental ingestion of feces and urine that contains S. typhi through dirty hands or contaminated food.

3. Symptoms include fever, rash (rose-coloured spots) and weakness.

4. S. typhi is excreted in the urine and stool

v. Rocky Mountain Spotted Fever

Rickettsia rickettsii is the causative agent of Rocky Mountain Spotted Fever

1. The disease is transmitted through ticks commonly found on dogs and cats and since the old man has a habit of feeding stray dogs and cats they may have transferred infected fleas to him.

2. Symptoms include high fever, headache, muscle pain, chills, malaise and rashes.

Tests Done

Preliminary tests done were general tests such as a full blood count (FBC), blood and stool cultures and biopsy of the rash.

Some of the confirmatory tests that are required to finalise our prognosis includes making specific cultures and also running various antibody-antigen reaction tests that varies in methodology.