Hello all!
Its my turn to share my SIP with you all Finally! Attachment is rather fun here and yup, i had learned alot of things from the staff working here. Till now, it has been a good experience. =)
In this hospital, there are alot of department. But for the 1st month of my attachment here, i am attached to the microbiology department, so basically what they do here is to culture and/or use different test to identify the pathogens that are causing infections. In this post, i shall describe one interesting reaction i saw in antibiotic susceptiblity test(which all of us should be very familar to). Susceptiblity test is done here to allow the doctors to know what antibioic to use base on the sensitivity of the strain of the bacteria. The method they make use of is called CDS method(invented by a group of people) to obtain better and more accurate results and reaction.
The method is the same as the one we do in micro lab. (See below)
Image taken from http://web.med.unsw.edu.au/cdstest/GTF_CDS_site/WebPages/HomeLevel/ManualFrames.htm
Usually after a patient's samples being cultured on agar shows significant isolates, it will be tested against beta-lactam antibiotics. Usually, Escherichia coli and Klebsiella especially can be resistant to these anti-biotics.
Beta-lactamase enzyme is a common bacteria resistance mechanism that is able to break down beta-lactam ring of penicillin-like drugs. They are able to hydrolyse antibiotics like CTX, AMC and CAZ. What lactamase enzymes do is that they break down the structural ring and make the molecules's anti-bacteria properties no longer functional. Therefore, when key-hole reaction can be seen, it shows that this bacteria strain is resistant to these antibiotics. The expression of the enzyme differs depending on the genetic control of the beta-lactamase production. TEM-1, TEM 2 and SHV-1 are the common plasmid-encoded enzymes that are resistant to penicillin.
The picture above shows Enterobacter being resistant.
So that is about all for this post. Hope that it is not too boring. Smiles =)
Zhenling TG02
Picture 3: Shows a general overview of how the virus can infect the cell and causes cell lysis. When the cells lysed, it forms a clearing in the plaquing medium. Each clearing can usually be visualized with the naked eyes or using some other techniques such as staining, microscopy, hemadsorption or immunofluorescence. Stain agarose is shown in picture 1 (above)http://www.flutrackers.com/forum/attachment.php?attachmentid=326&d=1148840493
Picture 2: Schematic diagram of plaque assay.Retrieved from http://homepages.strath.ac.uk/~dfs99109/BB211/phageinfect.jpg
Picture 1: Shows a Plaque assay on a 6 well plate. Agarose (or also known as plaquing medium) has been stained with neutral red for easy visualization of plaques. Staining is optional and our lab usually don’t stain it, we just check the number of plaques by holding the plate against the light to check for clearings/plaques.
Retrieved from: http://pathmicro.med.sc.edu/mhunt/plaque.jpg
Thanks for reading my entry =) very sorry for the late reply also, didn’t check that there were comments posted for my entry.
1) The type of mammalian cells I’m using is fibroblast but I’m not sure if I can tell you the strain. Sorry about that.
2) The cells that contain the gene I want from step 1 are actually from a company called invitrogen. The purpose of culturing these cells is only to get the gene which was supplied to us as cell cultures, because only these cells is known to contain such genes. The medium we use is according to what was stated in the protocol supplied by the manufacturer. Which was to use normal DMEM with 10% FBS supplemented. I believed the DMEM is a complete medium which already contains essential amino acids, vitamins and salts. Essential amino acids (i.e. those synthesized by the body) are required by most of the culture cells together with cysteine and tyrosine. However, this requirement will vary from one type of cell to another. Probably the amino acids in the DMEM is already sufficient. (I’m not sure but I will confirm with my mentor.)
As for antibiotic, the common and original use of antibiotics is to reduce the frequency of contamination. However, since we are already handling most of our medium in the LAF (laminar air flow hood) and BSC (Biosafety cabinet), there is no need to add antibiotics. We chose not to add antibiotic because the presence of antibiotic in the medium may “hide/mask” the presence of low level contaminations such as bacteria. If there is contamination, we would want to detect it and not use that batch of cultured cells.
3) I do not know why. LOL. =X And my mentor is not around to answer me also. (I will confirm with him when he’s back). Hmmz, but I do have my own answer to this questions. I think the virus titer drops over time because it is being stored in the cold room and used quite often. Although we wrap the tubes with the dull side of the aluminium foil to prevent exposure of the viruses to light (because they are sensitive to light), the opening of the cap still exposes it to light. As for the temperature, I really don’t know. =)
4) Well, he did test the virus efficiency earlier on when he first got the batch of virus produced, but the virus titer can drop over time and we may need to measure it again after about 3-4 weeks. For more detailed understanding of how the lab works read below =)
Batch 1 we call it P1, batch 2 we call it P2, and batch 3 we call it P3 etc. The very first batch of virus we get by following the outline given in the blog is called batch 1 or P1. This batch of virus titer is quite low and if we want to increase/ amplify the titer, we use P1 virus to infect a newly cultured 6 well plate of cells and harvest the virus. This is known as P2 viruses. P2 is supposedly quite concentrated and we do not use this P2 directly. Instead, we do amplify another batch from P2, called P3. This P3 virus is the virus that will be kept in the cold room and used frequently.
We have to produce new P3 stock once P2 virus titer is low. P3 can be used for quite a long time as it has about 20 plus ml, and usually we only need a few hundred µl. Whenever we get a new batch of virus, be it P1, P2 or P3 we will do a plaque assay to get an estimate of the virus titer and then these virus are kept back in the cold room. Without plaque assay we can not know the virus titer.(it takes about 7 days for the results of the plaque assay to be out). Also, the virus titer can drop over time, but sometimes we may have already used the viruses for some of our experiments and if we realized that we did not get the expected results, we may troubleshoot and do plaque assays to check if the virus titer has dropped. However, plaque assay requires quite a lot of cells and since our whole lab share only one flask of cells, maintained in a spinner flask, we can only do plaque assays when we have cells. These cells will only be split on Monday, Wednesday and Friday.
5) Well, since my mentor has dealt with this type of cells before, he already knows roughly the doubling time of the cells. Moreover, the manufacturers who provided the cells also provided the protocol that we should incubate roughly half to one hour to get 50% confluency. The confluency of the cells can be checked under the inverted microscope. 50% confluency means that the cells occupies about 50% of each well of the 6 well plate surface. If it is not 50% confluence yet, we can always return it back into the incubator for the cells to continue to grow. Usually, it should not take more than 1 hour to get 50% confluency for this particular type of cell we are using.
6) Well, the goggle is actually not necessary for the handling of the viruses. We handle the virus in the BSC and should not come into contact with our eyes. The goggle is actually given by the safety department for everyone working in the lab and is recommended that we wear it when we handle with large bottle of chemicals/ liquids, probably TAE buffer when preparing the agarose gel, because of the potential spillage. So I wear my goggles when I am preparing the agarose gel for running my sample. The goggles is required also when we are doing bench work that is handling with a lot of eppendorf tubes, the water droplets on the inner side of the cap may accidentally come into contact with the eyes as we pop open the cap. Also, since I do not wear my specs in the lab, goggles is required.
Jean Leong
TG02
Thanks for reading my entry =)
Plaques are visible structures (usually a clearing as seen in the picture 1) formed in a cell culture contained within a nutrient medium. (e.g the plaquing medium). These clearings has no cells in it because the virus infect the cell and eventually lysing it. (lytic cycle shown in picture 3). The cells that were initially seeded in the well and allow the virus to infect it (from step 1 to 4), then overlay with plaquing medium for the cells to attach to the agarose and grow. (if you use bacteria then a bacteria lawn will be formed;but a different method is used, on agar plate instead of 6 well plate. Done Before in Basic Microbiology. However we do not use bacteria in this case). Just like how each colony is derived from a single E.coli cell in an agar plate, each clearing here is derived from one virus attaching to one cell, and when the virus infects the cell and multiple it continues to lyse the other cells around and forms a clearing around that area. As that clearing, also known as plaque, arise from one virus, we are able to determine the number of viruses (which is also known as titer which is also = no. of plaque forming units/ ml) by counting the number of plaques. =)
1) Harvest the cells that the virus can infect on the 6 well plates and incubate til cells are 50% confluence.
2) Perform serial dilution of the virus such as 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8. in eppendorf tubes.
3) Remove medium from the cultured cells and add the diluted virus to the 6 wells and incubate to allow viruses to infect cells.
4) Remove the fluid (containing virus) in each of the well, as the viruses has already attached to the cells.
5) Add plaquing medium, which is a type of agarose gel containing medium to each of the well after the fluid is removed, and allow agarose gel to harden before incubating the plate of cells.
6) The function of the agarose gel is to immobilize the viruses so that it forms a clearing as it infects the cells.
7) These clearings (plaques) can be counted as plaque forming unit/ml, which is the titer of the virus.
(Picture taken from http://www.nalgenelabware.com/products/productList.asp?search=3&category_id=135&brand_name=Labware&category_name=Carboys)
Subject Title: Lab Techniques
Hi, Everyone. I am the fourth to post here and I am posted to a research lab. In the lab, there is a lot more chemistry processes than biology.
The lab I am attached to deals with phytoplankton…..a single-cell organism that can photosynthesize.
For this first post, I will be discussing on these:
1)Preparation of culture
2)Solid Phase Extraction (SPE)
The rest of the steps shall be discussed in future posts =P
For the preparation of culture, several carboys of sterilized seawater from an institute on St. John Island were collected personally. (take a ferry there =D). Why the seawater is collected from there and not other places? My supervisor say because seawater at that place is much more clean….less pollution. The seawater has been filtered through several different size filters and UV-sterilised at the institute.
Nutrients are added according to a given secret recipe.
The medium is then filtered using a filter unit.
The type of filtration carried out is pressure filtration.
This means gas is used to force the content from a cylinder tank to pass through the filter unit. The gas will travel from the cylinder, enter the pressure cylinder tank and force the medium from the tank to pass through the filter unit.
This is then followed by the addition of the seed cells. Then, the cells are left to grow in optimal conditions-optimal light intensity and optimal temperature.
After growing, we harvest the culture by separating the medium (supernatant) and cells (pellet) by centrifugation.
Time to harvest depends on the growth rate of the cells…….there is no standard amount of time to grow the cells…….if a lot of cells die then it is time to harvest.
The supernatant is then filtered.
Then SPE (solid phase extraction) is carried out on the filtered supernatant.
In the lab I am working in, the SPE used is reversed phase SPE. Other types of SPE include normal phase and ion-exchange SPE. I am not very sure about these others types of SPE since only reversed phase is carried out in our lab. Reversed phase SPE extract compounds base on polarity.
Reversed phase SPE use cartridges that contains C18 silica-based sorbent (stationary phase) which retain compounds by hydrophobic interaction. C18 means the silica is linked by a link that consists of 18 carbon atoms.
So, these cartridges are placed on top of a tank.
Picture taken from www.biotage.com/Print.aspx?id=35833)
1)Then, methanol (organic solvent) is loaded first to remove any impurities in the cartridges. Next, DI water is passed through these cartridges to wash away the methanol otherwise the desired compounds cannot be retained on the sorbent.
2)Then, the sample is passed through the cartridges, and the compounds will be bounded onto the sorbent by hydrophobic interaction.
3)DI water is then used to wash away impurities that may be present in the medium. It must be ensured that no DI water is left in the cartridges after washing otherwise it will inhibit the elution step.
4)To get the compounds from the sorbent, methanol is used as solvent to elute the compounds. The methanol will disrupt the hydrophobic interaction resulting in the compounds to detach from the sorbent and flow out of the cartridges with the methanol.
Actions carried out on the eluent and how to get the compounds from the eluent shall be explained in future posts. =)
Since, throughout the SIP, I will be dealing with a lot of methanol, PPE such as goggle, lab coat, and gloves are necessary. Also, the experiments are carried out in fume hood whenever possible. (Methanol is toxic when inhaled/consumed and can cause blindness.)
Hope everyone is enjoying your SIP. ^_^
Lim Xin Ni (0607325H)
TG02
Group 9
RSV under microscope( 20x)Picture courtesy of Molecular Micro lab(KKWCH)
Hihihihi! This is the 3rd week of SIP and its my turn to post! Hope all of you guys are enjoying your SIP!
Well I’m attached to the Microbiology department for 4 weeks. It has 2 laboratories, Microbiology itself and Molecular microbiology. One easy and interesting method we learnt back in school to detect virus/bacteria is immunofluourescence. I had the opportunity to practice it daily in Molecular microbiology lab.
In the lab I was attached to, tests were mostly done to detect respiratory viruses in children and infants. Such viruses include Respiratory Syncytial Virus, commonly known as RSV, Adenovirus, Influenza and Parainfluenza. Among these, RSV is considered the most common virus affecting children, especially below the age of 6. It rarely affects adults. RSV infects the lungs and the breathing passages causing wheezing, whooping cough and other breathing difficulties in children.
Usually when the doctor diagnoses pneumonia, bronchitis, bronchiolitis or other respiratory illnesses, they would send for tests to detect what virus has affected the child.
Samples sent to the lab are nasopharyngeal swabs or aspirates. Most common ones are swabs.
This is how it is being processed:
1.Firstly, samples should be checked with patient’s test request form to confirm that the identity is similar to the sample.
2.About 0.5-1.0ml of PBS should be added into a clear tube and the swab is placed into the tube.
3.A wire cuter is being used to snip the swab into the tube and capped. It is then vortexes until the suspension becomes turbid enough to be placed on the slide.
4.The slide is contains small wells and 2 wells are allocated for 1 sample. 1 drop in being placed on each well. This step is quite crucial as the size of the drop depends on the turbidity of the cell suspension. Have to make sure the size of the drop is as similar as possible on each well.
5.The slides should be air dried and fixed in cold acetone for 10 mines. It should be air dried after the slide is taken out from acetone.
6.10 µl of respiratory screen reagent should be added to one well. The respiratory reagent contains the seasonal reagent to detect the respiratory viruses.( RSV, influenza, Para influenza and adenovirus)
7.10 µl of RSV reagent should be added to the other well to detect for RSV as it is the most common virus affecting the children.
8.Slides should be incubated at 35-37 degree Celsius for 15-30 mins. After incubation slides should be rinsed with PBS for 5 mins to wash off the screening dyes.
9.They should be air dried and mounting fluid should be added followed by cover slips to be read under microscope.
10.If respiratory screen turns out positive and not the seasonal viruses, then the procedure is repeated again.
How to read the slides and recognize the viruses?
It is quite beautiful to see the slides under microscope. In fact, they are quite glaring when they turn out to be positive. Positive results mean that the cells fluoresce, which indicates that the patient is infected by that virus. Usually if the screening reagent and the RSV turn out to be positive, it proves that that patient is infected by RSV. However, if RSV turns out to be negative and screening reagent turns out to be positive, it means that the patient is infected by other viruses. It could be predicted by the morphology of the cells.
Parainfluenza would have a granular cytoplasm and the cells would look a little ‘bloated’.
Influenza virus would have a green cytoplasm.
RSV would have a crescent like shape at the edge of the nucleus.
These are the typical characteristics of the cells. However, this is not the case in all situations. Sometimes, it very difficult to identify them unless you are very used to looking at them
Hence, most of the time confirmatory screening has to be done using the specific screening reagents (like influenza, parainfluenza or adenovirus). If Influenza or parainfluenza is being identified, they are being sub grouped into influenza A or B, Parainfluenza 1,2 or 3.
So this is how immunofluourescence is done in Molecular microbiology! I know it’s quite lengthy, but sorry, I have to add all the details. Have fun!
BY NEELA
TG 02
References
http://kidshealth.org/parent/infections/lung/rsv.html
Sorry to all. To view the picture, please visit the website given.
Sorry for the inconvenience caused. =)
Debbie.
Hmm.. i'll try again.
Picture taken from:
HI all, sorry for the late submission, computer was down and all. In any case, for my first 2 weeks, I was posted to the Urinalysis/ Routine section. As most of the tests done are through automation, I was required to learn how to operate the machines, the quality control tests made at the start of the day or at specfic hour of the day. There were times when the urine FEME tests are required to be done manually using dipsticks due to insufficient amount of sample. My job was to load up the specimen into a tube, ensuring that it has been labeled correctly before being placed in a rack for loading into the Atlas machine.
Picture taken from:
Clinitek Atlas is a fully automated reflectance spectrophotometer used to perform urinalysis testing. The various results retrieved are the glucose level, bilirubin, ketone, specfic gravity, blood, pH, protein, urobilinogen, nitrie. Leukocytes, the color and the clarity of the urine. It is capable of performing up to 200 samples at one go and that each roll is made up of 490 reagent strips. The samples(minimum volume ≥ 5ml) are loaded onto the sampler tray (maximum 10 tubes) and placed onto the infeed area.
Firstly, the bar code reader would scan the sticker on the tube for identification purposes and the automatic pipette will come forth to collect the specimen via the syringe pump and then dispenses a specific amount of sample onto each of the reagent strip pads and into the SG well for determination of the specific gravity and clarity. The remaining sample are dispensed into the rinse well followed by a larger volume of rinse solution that rinses both the inside and outside of the pipette. Finally, the pipette dispenses the rinse solution into the SG well to ensure complete rinsing of the well.
Quality controls are done every morning by the morning shifts. The CLINITEK ATLAS Control solution(s) are prepared by soaking CLINITEK ATLAS positive and negative control strips in 12ml of deionised water each for 30 minutes before being run under ‘ Control analyze mode’. The results should tally with the reference range provided by bayer healthcare.
Yup, that’s all for now. Any questions, feel free to ask me. Haha. I will do my best to answer them. =)
Debbie.
Hi Johan, thanks for reading my entry.
The method we use to transform the cells is: Heat Shock at 42 degree Celcius
This is how we do it:
1) Use 50-100µl of competent cells (E.coli), and add 0.5- 1µl of recombinant vector and incubate tube on ice for ½ hour. (keep the cells cold so that transformation efficiency will remain high).
2) Warm up agar plate that has amphicillin antibiotic, in a 37 degree Celcius incubator
3) Ensure water bath is warm up to 42 degree Celcius, ready for the heat shock process. (heat shock the cells so they open up their pores and DNA enters)
4) After ½ hour incubation, heat shock the mixture in step 1 for 45s in 42 degree celcius water bath, and immediately transfer it onto ice for 1 to 2 mins. (cool it immediately so that the pores closes back).
5) Add S.O.C medium (something similar to LB broth) in a 1:9 ratio. E.g. 50µl cells: 450µl of S.O.C .
6) After adding the S.O.C medium, put into 37 degree celcius incubator with shaker for 1 hour.
7) Obtain the transformed cells form the incubator, do a 50x dilution and plate it on the amphicilin plate.
8) Do 2 spread plate, one with the 50x dilution and an undiluted spread plate.
9) Allow both plates to stand for 1-2 mins then invert the plate and incubate it at 37 degree celcius for 1 day.
10) The following day, observe for any colonies formed. These cells are resistant to amphicilin, because of the amphicilin gene in the vector, acquired through the transformation process.
Blue White selection strategy (use to determine the presence of insert)
The blue white selection strategy makes use of alpha-complementation, which means the alpha and the beta part of beta-galactosidase is “completed”.
The diagram (above above) (duno why it appear at the top) is a lac-operon: lac operon is only switched on when glucose is absent and lactose is present.
IPTG (analog of lactose) is a gratuitous inducer of the lac operon.
Grow the colonies in the presence of X-gal, which is a colourless compound that can be cleaved by ß- galactosidase to produce blue colouration.
The alpha and the omega peptide must be “completed” for ß- galactosidase to be functional to cleave X-gal.
The lac Z alpha gene is found in the vector, and only when the vector is introduced into the lac Z alpha mutant (no alpha peptide) during the process of transformation, then there will be alpha complementation and produce functional ß- galactosidase and cleave X-gal to produce blue coloration. (Blue Colonies).
However, if there is an insert in the vector, because of successful ligation, the presence of the insert will cause disruption to the lac Z alpha gene and there will not be alpha complemention > no functional ß- galactosidase> no cleaving of X-gal >colonies remain white.