Sunday, July 29, 2007

Microbiology and Accreditation of Labs

Hi all. It’s nice meeting everyone again. You people must have missed school a lot. I'm currently doing in house SIP. So far, I'm spending most of my time doing literature review. I do not really have interesting things to share because I’m just looking at different protocols for food analysis and doing a little read up on accreditation of labs. So pardon me if this entry gets boring. Unlike many others, I do not have to deal with machines. Things that I’ve done require basic skills only.

For my SIP, I am a TSO for Basic Microbiology as well as Human Anatomy and Physiology practical lessons. For B Mic, i did streak plate for Bacillus Subtilis, subcultured of E. coli on nutrient agar and broth. Because of the shortage of slides for viewing, I had to do gram staining for S. aureus, E. coli and Bacillus Subtilis. All the slides were mount using DPX to preserve the stains. I also prepare agar plates and dry agar plates. Agars were prepared by weighing the dehydrated media and suspend them in distilled water. Molten agar is then sterilized by autoclaving at 121°C for 15 mins.

As for HAP practical, I prepare the required reagent or equipment required for that day’s practical. Boiled starch, pepsin, pancreatin, egg albumin, litmus milk were prepared and dispensed into the bottles.

As a TSO, there are also some general duties for me to do. Housekeeping, clearing the oven, refilling the DI water, ethanol as well as washing up are some of the things expected to be done every week.

What I’ve done in AS4 is microbial food analysis testing. This is done by using AOAC official methods to find out the total plate count, coliform count and E. coli count. The test selected is 989.10 (total plate and coliform) and 991.14 (E. coli). The food sample used is different flavours of ice cream. The steps for performing this test are quite straight forward.

First, dilution of ice cream is done by weighing a certain required amount into Butterfield’s buffered Phosphate diluent. Mix well. Inoculate homogenate onto petrifilm. Incubate the petrifilm. Read the results.

The picture above is an example of one of the results. This result is for chocolate ice cream. 1 and 2 are results for TPC, 3 for CC and 4 for EC. For CC, only red colonies with air bubbles next to the colonies will be counted. The counting range for TPC, CC and EC are 25 to 250 colonies. Anything below 25 will not be counted and anything above 250 will be reported as estimated count. In this case, of all the 36 petrifilms, only 6 had readings, whereas the rest were not counted due to the low amount of colonies growing on the petrifilms. (so the ice cream are still consumable. this is considered low as compared to char kway teow. i heard the microbial count for the latter could be more than 10X more.)

To get a lab accreditated, certain standards have to be met. Some of which include validity of agar, microbial count of water, sterility of autoclaved bottles. I did a trial test for validity of agar by streaking microbes (E. coli and S. aureus) on general agar (TSA) as well as selective agar (MacConkey agar and Baird Parker agar). This is to ensure that the agar is correctly enhancing and inhibiting the growth of the different microbes.

Also, I did microbial testing of water. Choices of water used were milliQ water, DI water from RO tank, DI water from container and tap water. The results are as follow

Pic 1 and 2: milliQ water, Pic 3 and 4: DI water from RO tank, Pic 5 and 6: DI water from container, Pic 7 and 8: Tap water

Since microbial food analysis is involved, it is very important that test or experiment is done in a controlled environment. There must be a microbial quality monitoring program for the laboratory environment as well as the de-ionized water used in the laboratory. This is done by establishing and verifying suitable and effective protocols based on standards methods to measure microbial bioburden in the air, work surfaces, and de-ionized water. Using the data collected, statistical analysis may be applied to determine the monitoring frequency, alert and action limits, etc, all of which will contribute to an effective monitoring program.

15 weeks of SIP left. All the best to everyone. enjoy. =)

Suat Fang

Tuesday, July 24, 2007

Laboratory Techniques

Hi all, Royston here. How are all of you at work? Sorry for not posting before the weekends, because I left my thumbdrive in school locker. For these past 4 weeks, I have been doing literature searchs, protocol write-up as well as subculturing Human Umbilical Vein Endothelial Cells (HUVECs) for my major project.

From the literature searchs, I have learnt that atherosclerosis is actually a chronic inflammatory disease. Cell – cell interaction between circulating monocytes and vascular endothelial cells is a key event in the initiation of atherogenesis. Recent studies have shown endothelial cells treated with soy proteins of isoflavone class inhibited monocyte adhesion, thereby suggesting the athero-protective effects of soy proteins.

I have written protocols of cryopreservation, feeding, and gelatin coating for culture dishes, subculturing and thawing of cells. Protocols are required to minimize the occurrence for errors as well as for trouble-shooting purposes.

Human Umbilical Vein Endothelial Cells (HUVECs) used for my major project, were purchased from Amercian Type Cell Collection. The main advantage of using HUVECs to study endothelial biology is the wide availability of the umbilical cord, a relatively simple method of isolation and the general purity of the cell population obtained. It is important to note that, as with isolation of any cells from a human tissue, there is a potential of risk of infection. Precautions for working with human tissues, such as wearing gloves, a laboratory coat, and safety goggles, must be used at all times. Working in a Biosafety Laboratory level 2 (BSL-2) is also required. This laboratory is negatively pressurized by a vacuum with an ante room in front of the lab, in which these 2 features do not allow any contaminated air from leaving the lab.

1 vial containing 1 mL of HUVECs was thawed and plated on 100-mm 0.2% gelatin coated dishes in commercially prepared HUVEC medium. After 1 day of incubation, media change is done to dilute the traces of DMSO, which is toxic to cells, used in cell freezing media during cryopreservation. After 2 days of incubation, feeding (media renewal) is done to replenish the growth factors and supplements in the media as well as removing any metabolic waste or dead cells that may be inhibitory to cell growth.

However, the picture above shows the cells morphology before feeding, the cells shows severe signs of deterioration which includes granularity around nucleus, cytoplasmic vacuolation and rounding up of cells with detachment from sustrate.

Based on the picture above, it can be seen that all the cells have become unhealthy as are fractile particles present within cells. Cells have slow cell proliferation. Troubleshooting is done to determine the cause of the problem and improvise solutions to solve the problem. Cause was identified as seeding density too low. When HUVECs are set up at too low a density, there is minimum cell-cell interaction between cells. This results in formation of unhealthy cells and slow proliferation. By passaging the culture frequently in new fresh media will solve the problem. This causes the unhealthy cells to die off eventually while new healthy cells will be generated via cell division in fresh medium with abundant cell growth factors.

Picture above shows cells after 3 days of incubation in fresh medium, another round of feeding is required to constantly provide growth supplements for continuous cell growth. The cells are actively dividing and proliferating. A subculture is required once confluence of the dish is reached.

Royston Tan

Monday, July 16, 2007

LMQA- Ordering tests

Hi all!

Hope you are all enjoying your SIP thus far. I am learning quite a lot already and am finding it quite hard to keep all the information I've been given into my brain. =D

So I'm attached to a clinical laboratory of a really pretty hospital> It is a rather small laboratory which is divided into different stations like Haematology, Blood Banking, Chemistry, Cytology, Microbiology, Urinalysis and Order Entry 1 and 2.

For the first 2 weeks, I was stationed to Order Entry 1. This is after I was told to read 3 thick files of safety manuals, spills and fire safety and general policy manuals. This proved to be useful because the manuals explained in general the roles of the staff and even how some of the machines work.

Specimens are send to the lab via a pneumatic system at the Order Entry 1 (O1) station. This cuts the travelling time taken for the nurses or porter to personally send the specimens to the laboratory. Once the specimens reach the lab, the requisition forms are scanned into the LIS in the computer at O1. The forms must contain the following:
  1. The patient's name and IC number and his demographics
  2. The location of the patient (ie ward/clinic/others)
  3. Name of requesting doctor
  4. Whether it is urgent
  5. What tests are being ordered
It is also the medical technologist's stationed responsibility to make sure that the type of blood tubes sent corresponds to the tests ordered. For example, routine tests such as FBC requires an EDTA tube which is indicated with a lavender top. If no EDTA tube is sent with an requisition form ordering EDTA, the med tech must call the nurse-in-charge and address the problem. As mentioned above, the blood tubes are colour-coded to represent the type of blood sample.

Blue top- Citrated blood, Red top- Plain blood, Lavender top- EDTA blood, Green top- Heparin Blood, Grey top- Fluoride & Gold Top- Gel tube

Blood is not the only samples that are being processed in the lab. There are also blood cultures, stool samples, sputum samples, gastric aspirate and urine samples. Blood tubes are sent to the chemistry, haematology and blood banking station, depending on what tests are ordered. Some urine samples (depending on what test is being ordered) are sent to the chemistry station. Blood cultures, stool samples and sputum are sent to the microbiology station. 24 hours urine are sent to the urinalysis station.

Ocasionally there will be a need to reject certain samples. However, before rejecting a test, a confirmation with the nurse-in-charge of the patient must be made. Afterwhich, the name of the staff informed must be recorded into the LIS so that traceability can be acheived. Here are some of the grounds by which the order must be rejected:
  1. Specimen sent without requisition form
  2. Insufficient sample (particularly blood tubes)
  3. Unlabelled or incorrectly labelled sample
  4. Broken tubes
  5. Specimen not sent in ice ( eg iced heparin syringe for Arterial Blood Gases test and iced EDTA tube for renin test)
  6. Incorrect blood tube sent for order
  7. Clotted blood
Well, although this is very routine and all, it is the most important part of the whole process. If the test is ordered wrongly it will affect the overall turnaround time (TAT). The laboratory has a specific tunaround time for STAT and routine tests of which it hopes to acheive or improve every month. As such, it is important that test that are meant to be rejected are not ordered so as to minimise the tunraround time.


Saturday, July 07, 2007

Lab Techniques and Microbiology work

ok! This week is my turn to blog what I had done for the past weeks..

Well, I am doing my SIP in house and during the first week there is nothing much to blog about as our TSO in-charge was not assigned to us yet and also, I wasn't quite sure about what to do for my MP yet..So basically, during the first week, I just tagged along with my partner, observing what she was doing and taking notes at the same time. On my own, I did subculturing of human lung cancer cells. This is carried out in the BSL lab of AS4. The laminar flow hood that was use was of class 2 category as human lung cancer cells ( NCI-H460) or known to us as 177 was rather pathogenic, thus proper preventive measures had to be taken care of. The laminar flow hood that I used has a alarm system, and it will sound if the shield is raised above the protective level to inform the user to lower the shield down for personal protection purposes. Subculturing is done when the cells had reach about 60% confluency. Confluency refers to the space that the cells had occupied, and the cells viality will decrease with increase confluency, thus it is crucial to monitor the cells' growth under the inverted microscope and carry out subculturing when required. Trypsin is required to help digest the cells to enable the cells to be detached from the flask surface. After which, media is added to the flask containing the trypsin and cells to help stop the trypsin action, thereby preventing excessive digestion of the cancerous cells.

Apart from subculturing, I was also introduced to the various machines preset in the labs and one of them is the Beckmen Avanti J251 Centrifuge, also known as the standing centrifuge. This centrifuge is able to centrifuge at a very high speed of about 20,000 rpm or even higher. One very troublesome about this machine is that it is balanced based on weight and thus before putting the centrifuge bottles into the centrifuge, it is a must to measure the bottles and the difference of each pair can only be +/- 0.05g. Anything more than this tolerance will cause imbalance and the centrifuge will vibrate. Similar to the usual centrifuge, the bottles of similar weight are placed opposite one another to allow balancing. The bottles used are also round bottom as conical flasks or others non-round bottom flask might break and result in spillage.

As my project requires herb extraction, I had to make use of the rotary evaporator to enable the evaporation of the 50% ethanol in which my herb ( S. barbata) is dissolved in. This evaporator allows the round bottom flask to be rotated when it is being heated so as to enable uniform heating and evaporation. Connected to the heater is also a condenser and vacuum pump. The condenser is there to allow the evaporated ethanol to condense and fall into the other attached flask and the condensed ethanol is labeled as waste and thrown into the waste bottle. The vacuum pump will enable heating to be carried out at a lower temperature of about 35 degrees. At this temperature, ethanol (boiling pt of 78 degress) is usually unable to evaporate so quickly, thus the vacuum can lower the boiling point and quicken the heating procedure.

During the 2nd week, I was already assigned to a TSO and had to do pour plate of Potato Dextrose Agar (PDA) and Nutrient Agar (NA). At first I thought a pipette had to be use to aliquot the agar into the culture dish, but my supervisor told me that all I had to do was to pour the agar from the bottle into the dishes. This is because, by using a pipette, there will be alot of unwanted bubbles present in the agar and it will affect the state of the agar. When I first poured, it was relatively difficult to estimate the required volume so as to get the agar of the best thickness, and i spilled some of the agar. However, after some practise, it became easier. In addition to that, I was told to prepare 5L of NA. This is done by dissolving 23 g of NA powder in 1L of DI water. Its just as simple as that. Then the 5 bottles was autoclaved.

These are basically what I had experienced for these 2 weeks. Nothing much, mainly just some operating of the big machines which I thought was complicated but doesn't turn out to be as difficult as I thought it was, and the ongoing subculturing of cells. Feel free to ask any questions and hopefully I am able to answer all of them.

Have Fun during your SIP guys! Take Care!

TG 01

Sunday, July 01, 2007

Attachment Experience

hello guys (:

In case if you're wondering whoose the first to blog, its me! Natalie.
So, i shall get the ball start rolling.
This is the first week which i was posted to this hospital for attachment. Basically, our lab consists of severals sections namely, immunology, processing, microbiology, histology, bloodbank, biochemistry etc. For the first two weeks, i was posted to the Biochemistry Lab.

The biochemistry lab mainly handles patients' serum or urine samples for testing of analytes or electrolytes e.g. glucose, potassium, magnesium, albumin, ALP, ALT, CK, CKMB, Pro BNP etc.
However, not all testings are done by a machine alone. There are several machines used in the lab for different kind of testings.
For example,
Test for Renal and Liver Panel, Lipid Profile and individuals test like, albumin, ALP, ALT, Phosphate, Magnesium is used by the machine, Beckerman Coulter's; LX 1 & 2.
Test for CKMB Mass, Troponin T are used by the machine, Elecsys.
The other machine Cobas, is used to test for PSA etc.

So what actually happens before we load the plain tube contained the patient's serum into the specific machine, afterwhich we retrieve the result from the computer and print them?
Firstly, a blood sample is collected from the patient. The blood sample is then sent to the processing counter whereby labelling of the patient's identity as well as the type of test to be done is made.
Then it is being centrifuged at a certain speed for 5minutes, afterwhich, i'll collect them and seperate them according to which type of test that specific tube is meant for and load then into the specific machine. Its as simple as that!

However, in such cases whereby the serum is insufficient, we have to transfer them into smaller cups before loading them into the machine. It is becase the machine only can retrieve the serum to a certain level. Any amount lower than that level, it will be posted on the the screen as 'probe obstructed' etc. Addition to it, the machine will rings if any other problems occurred.

On the third and fourth day, i was asked to arrive there earlier to observe and learn how the supervisor and staffs there operate the machine. Every morning, it is a must to check whether there is sufficient reagents to carry out the various test. And we also have to check whether has the calibration of each and every reagent has expired. It is crucial because if ever so, there is insufficient reagent or expired ones, we are unable to use the machine and it will decrease the turnover rate and slow down everything. It could do harm especially to those who are under the awaiting(emergency) list. Furthermore, we ought to carry out controls whenever we have entered a new reagent into the machine, which take time to process too.

Lastly, i also have learnt that in this hospital, wards starting with the number 3,4,5 e.g. 23,44,35 are wards that are under the awaiting list. In another words, urgent cases whereby, we are supposed to send the results over within an hour. And one more thing, which is the most important thing to do before you process anything; you must always remember to check whether the patient's identity tallies with the tube that your're going to process with! It will be a disaster if wrong result is sent to the wrong patient. Not only you'll get blamed, the whole entire staffs involved will be pulled down too. So, remember to check properly before processing! (:

Urm, so i have kinda summarised everything that i've done for the past week in a nutshell (: By far, I am enjoying what i am doing and am glad to learn new stuffs everyday. The staffs there are friendly and approachable so, everything is fine for me (so far i guessed)! (:
Erm lastly, any questions you may post it in the text box? Hopefully, i'll have the answers. HA!
Hope to hear from you guys soon! Takecare!

Natalie Teo