The Miscellaneous Bench

Hello everybody! This is Jeremy Chu (TG01) attached to bacteriology posting on...

The Miscellaneous Bench

The miscellaneous bench deals with every other specimens besides blood, stool, urine and respiratory specimens. Fluid, tissues and swabs are mainly the samples received. Some examples are heart/liver tissues, peritoneal fluid or peritoneum fluid, wound swabs and high/low vaginal swabs. Detecting bacteria at these sites are important, especially for sterile sites. There should not be any bacteria found at sterile sites such as the heart and liver or patient’s situation could be fatal.

Wound cultures are performed to isolate and identify bacteria infections causing an infection of the wound. This way, doctors can then administer the appropriate antibiotics to kill the causative agent.

Fluid culture is done, similarly to isolate and identify bacteria causing an infection. Peritoneal/Pericardial fluids are sterile fluids and any presence of bacteria is regarded as significant. Gram stain is also performed.

High vaginal swabs are cultured for the purpose of detecting group B streptococci (commonly Streptococcus agalactiae) found in the female reproductive tract. S. agalactiae can cause pneumonia and meningitis commonly in neonates and the elderly.

The different types or media used and its purposes are listed in the table below.

Media	Purpose	Types of specimens
Blood Agar	Enriched media to enhance bacteria growth	Tissues Fluids Fluid swabs Wound swabs Catheter tips/devices Vaginal swabs
MacConkey Agar	Selective media to inhibit most gram positive bacteria and differentiates lactose fermenters
Blood Agar (anaerobic)	Enriched media to enhance anaerobic bacteria growth	Tissues Fluids Fluid swabs
Cooked Meat broth	Enriched broth for cultivating bacteria	Tissues Fluids Fluid swabs Wound swabs Catheter tips/devices
LIM broth	Enriched broth used to grow group B streptococci from vaginal swabs	Vaginal swabs

Fluid culture agar plates are incubated under CO₂ conditions, while its broths in aerobic conditions. Blood, MAC, blood (anaerobic) agars and cooked meat broth are the choice of media.

Blood agar and LIM broth are used for vaginal swabs, while blood, MAC and cooked meat are used for ordinary wound cultures.

Roche Cobas e601 Analyzer

HI EVERYBODY!
stella here to post another entry!
I'm going to talk about Roche Cobas e601 Analyzer.

As i'm attached to a lab with mostly automated machines, therefore most of the tests are run on analyzers.

Roche Cobas e601 analyzer run different kinds of tests such as cardiac markers (CKMB, myoglobin, NTproBNP, Troponin T), tumour markers (AFP, CEA, CA125, CA15-3, free PSA, Total PSA) and bone markers (beta cross laps and osteocalcin)

An example of a test that is run on the Cobas e601 analyzer are cardiac markers such as Troponin T.
The purpose of performing the test is as follow:
Troponin T is useful and important in the diagnosis of myocardial damage such as myocardial infarction,

Principles of the tests : Immunoassay with Electrochemiluminescence technology

During the assay, the sample containing Troponin T is incubated with two monoclonal antibodies specific to the Troponin T. Examples of the antibodies against Troponin T are biotinylated monoclonalTroponin T-specific Antibody and a monoclonal troponin T-specific antibody labeled with a ruthenium complex. Thus, a sandwich complex is formed. Afterwhich, microparticles coated with streptavidin are added and the complex will be bound to to the solid phase through the interaction of biotin and streptavidin. The entire complex are drawn onto the electrode magnetically, with unbound components being washed away.
Electrochemiluminscent is induced by the application of a defined voltage. The photomultiplier measures the emission of light produced.

Reference interval for Troponin T:
<0.03ug/L

Troponin T can be futher divided into skeletal Troponin T and cardiac Troponin T.
Cardiac troponin T is a specific cardiac marker used for the diagnosis of myocardial damage.
It has unique cardiac specificity as it has different amino acid sequenes which are different from that of skeletal troponin T.
The presence of cardiac Troponin T in the bloodstream indicates there is myocardial damage.
In a healthy individual, no levels of troponin T can be detected in the bloodstream.
troponin T is a better cardiac marker due to its ability to stay in the bloodstream up to 2 weeks.
It rises from 4 to 6 hours, peaks at 12 to 24 hours and persist in the blood for 2 weeks.

Oral Glucose Tolerance Test

Hello everybody!

This is Hakim for another round of blogging.

NB: This is an edited entry. I have not removed anything, but have added in a few more points.


PURPOSE OF OGTT
The oral glucose tolerance test (OGTT) is a test performed to determine if the patient has diabetes. In my lab, majority of the patients who undergo this test is pregnant. This is because pregnant women could acquire gestational diabetes, which is a form of type II diabetes that is first diagnosed or begins during pregnancy. This condition usually ends soon after the person gives birth.

In a nutshell, the patient would have to give a urine sample and have their blood drawn out. After this, the patient is given a large dose of glucose and subsequently, urine is collected and blood is drawn after a period of time (depending on the GP's request). After the final urine collection and blood drawing, the urine samples are tested for glucose and ketones by using the dipstick. The blood tubes are centrifuged and its plasma is put in an analyzer to test for glucose levels.

PRINCIPLE OF TESTING PLASMA GLUCOSE LEVELS
Our lab is using the COBAS Integra 400 plus. It uses the principles of light absorbance and colorimetry. Glucose in the plasma is oxidised by glucose oxidase, producing gluconic acid and hydrogen peroxide. Peroxidase converts the hydrogen peroxide into oxygen and water. The oxygen liberated in the reaction is taken up by 4-aminophenazone with phenol and forms a pink-coloured substance. The substance can be measured at a wavelength of 515mm. A halogen lamp will produce a light beam and passes the cuvette. A photodetector would sense the amount of light passed through the cuvette (and also the amount of light absorbed by the substance). It is important that calibration is done frequently so that the results would be accurate.

PROCEDURE OF OGTT
It is important to note that the patient is required to fast for 8 hours before the test commences. This is done so as to prevent any pre-analytical variations. For example, a high plasma glucose after a meal could misdiagnose the patient as having impaired glucose tolerance (IGT). The patient is allowed to drink water during the fasting, but they are advised to try to drink as little as possible as the blood could be diluted if they take in large amounts of water. Also, the tube collected is a fluoride tube so that it will reduce the rate of glycolysis by the red blood cells so as to stabilize the levels of glucose. So after the first round of specimen collection, the patient is given a 75 gram glucose drink to drink. She has to finish the whole bottle. It tastes like concentrated F&N Orange. From my own experience, the drink is extremely sweet. I couldn't even finish the whole bottle (which is about 350mL) so I'm not sure which is more painful for the pregnant woman; childbirth or finishing up the whole bottle. There are 2 different timeframes for blood to be drawn, depending on the GP's request. It's gonna be quite confusing, so let's give an example. - Patient A is requested to have her blood and urine collected 1 hour AND 2 hours after drinking the glucose - Patient B is requested to have her blood and urine collected only 2 hours after drinking the glucose So after both patients finish the bottle, she comes back in either 1 hour (for patient A) or 2 hours time (for patient B), depending on the GP's request. During this time, she still cannot eat or drink anything (other than water, but in small amounts). Patient A comes back in 1 hour to have her blood and urine collected. At 2 hours after drinking the glucose, BOTH Patients A & B comes back to have their blood and urine collected. Only after this is done, they are able to eat and drink. For urine sample: The dipsticks are dipped into the urine to check for glucose and ketone levels. A high level of glucose in the urine would mean that there is a high level of glucose in the patient's bloodstream and the levels have exceeded the kidney's threshold. So the kidneys aren't able to reabsorb any more glucose and is then excreted through the urine. Ketone bodies are a result of lipid metabolism. This is caused by starvation. Ketone bodies would be present in diabetics. For blood sample: The blood tubes are centrifuged. The blood tubes MUST be labeled (0 hr, 1 hr, 2 hr) so as to prevent any confusion. The tubes are then put in an analyzer machine. For our lab, we are using the COBAS Integra 400 plus.

Note: For some reason, I can't upload pictures, so here is the link to the website that contains the picture of the analyzer.
http://labsystems.roche.com/content/products/integra_400plus/introduction.html
REFERENCE RANGE OF OGTT If the levels of plasma glucose is higher than normal, then we can say that the pregnant woman is suffering from gestational diabetes. There are many diagnosing criteria for OGTT. The World Health Organization's (WHO) guidelines for diagnosing GDM is ≥5.3 mmol/L for 0-hour plasma glucose, OR ≥10.0 mmol/L for 1-hour plasma glucose, OR ≥8.6 mmol/L for 2-hour plasma glucose. However, some doctors do not request for a 1-hour plasma glucose because it is not as clinically significant as the 0-hour and 2-hour plasma glucose levels.

They are advised to maintain a healthy lifestyle and have a proper suitable diet. They can also take insulin.

Ok that's all from me. If you have any questions for me, I'd be glad to answer them as fast as possible.

Happy SIP-ing!
Hakim
0703555C

13 more weeks!!!

Handling, restraining, sexing and identification of rats and mice

Hi all. I am currently working in the Animal Facility for a particular instiitution which cannot be named due to issue of confidentiality. I am juggling both my major project and my SIP thus it requires alot of initiative and time management. I usually assist in lab practicals and the maintenance of the animal husbandry while carrying out my experiment at the same time.

I shall share with you the Handling and restraining of the rat/mouse,the sexing and identification of the rats/mice which is essential as these techniques form the basics before other procedures can be carried out.

Whenever animals are used for research or teaching, it is important to minimise pain and distress that they may suffer as this would indirectly in turn affect the reliability and reproducibility of the experimental data. AThe adoption of the 3R principles as required by NACLAR Guiding Principles, such as refinement of procedures, replacement of animals and reduction of the number of animals used should become an integral part of all scientific research

HANDLING AND RESTRAINING TECHNIQUES
The use of proper handling and restraint techniques would help to minimiza unneccessary stress to both animals and research personnel.Its is recommended that the animal be handled on a regular basis in a non-threatening manner such as weighing, giving food treats etc. Most animals will lrespond positively to learn to recognize individuals. Suden movements prior to handling the animals may startle them.

Handling and restraint of Mice

1. Removal from cage
WHen handling mice, your movement should be slow and gentle.
Grasp the middle or near base of the tail between your thumb and your index finger, and lift the mouse out of the cage. Never hold it by the end of its tail. The mouse may also be picked up using a thumb forcep with its tips covered with rubber or polyethelene tubing to prevent damage to the mouse tail. Do not apply too much pressure when lifting the mouse as this may cause the tail to break.

2. Technical manipulation
Mouse has to be placed on a rough surface in which it can firmly hang on to such as the cage lid. Smooth surfaces will frighten the mouse because it cannot get a foothold. Gradual pressure can be applied to the tail while the index finger and thumb are used to scruff the skin around the animal's shoulder.ATurn the animal over to rest in the palm of your hand. Its tail may be restrained by the other hand or simply between the last 2 fingers of the hand holding the mice. THis enable the other hand to be free to carry out manipulations or injections.

Handling and restraint of Rats

1.Removal from cage and Manipulation
THe rat may be lifted out of the cage by the base of the tail. Never suspend the ray by the tail for too long. SUpport the body weight as soon as possible so as to minimise stress to it. Gently and firmly grasp the rat around the thorax with thumb and fore finger under each of the front legs. The rat might be stretched out by pulling the hind legs with the other hand.

Identification of Rat and Mice

THere are various ways for identification of rats and mice
1. Cage card - to identify strain of rat/mouse, sex, number, principal investigator and research protocols. This should not be removed from the cage to prevent misidentification
2. Pen marks on tail- this serves as a form of temporary identification of an individual. THe marks usually last for 1-2 days long
3. Hair clippings or dyeing the fur - may last up to 14 days
4. Ear tagging - small metal clips stamped with individual numbers applied near base of ear
5. Ear Punching and notching - rats can be identified with number 1 through 99 by piercing a hole or a notch or a double notch or any combination. 1,2 and 3 are holes punched on the right ear while holes punched on the left ear represents 10,20,30 (i dunno how to post pix)
while 4,5,6 and 40,50,60 are represented by notch and 7,8,9 and 70,80,90 are represented by double notches.

Major Project: Identification of compounds in tea that possess anti-microbial properties against Staphylococccus aureus

Hello, I'm Dennis from TG01 and I'm currently in school to complete my Major Project within 10 weeks before I move on to do my SIP, reason being the hospital that I'm attached to didn't have MPs to assign us with. Let me give you a brief overview of my MP.

The aim of my MP is to identify compounds present in tea that possess anti-microbial properties against Staphylococcus aureus. This is achieved by using a whole cell-based assay to test for anti-microbial activity of tea, followed by using High Performance Liquid Chromatography (HPLC) to identify specific compounds.

The cell based-assay in this case is used to test the anti-microbial properties of tea against S. aureus at different concentrations. The assay is made up of a 96-well micotitre plate, consisting of positive control, negative control and as well as different concentrations of tea. Ampicillin is as a standard.

My MP is actually a continuation on the work of a previous student doing her MP last year. She has completed the optimisation of the whole cell-based assay. What I had to do what to carry out some replicate experiments to ensure there was reproducibility of data. After I have ascertained the results I've obtained is reproducible, I can carry on to testing different types of tea for anti-microbial properties. There are several parts to my MP, so I'll be only covering the steps that are involved to determine the IC50 (Inhibitory Concentraion @ 50%) of Ampicillin

Firstly, I had to obtain pure colonies of S. aureus, and this is done by using the streak plate method on Tryptic Soy Agar (TSA). After overnight incubation of 18hours @ 37 degrees celcius, I would then subculture a pure colony of S. aureus 10ml of Tryptic Soy Broth (TSB). Again, it is incubated overnight for 18hours, at 37 degrees celcius and shaking at 150rpm.

Next, I would take 1ml of the culture and subculture it into 49ml of TSB (1:50 dilution). I would then allow the cells to grow to the log phase at the 3rd hour. The log phase has been determined by doing some experiments prior to this, whereby an absorbance curve is plotted using data obtained from the spectrophotometer.

After incubation of 3 hours, I would then use a hemocytometer to obtain the cell density of the culture. The culture was then diluted to obtain a final cell density of 1 X 10^6 cfu/ml. After the final cell density is obtained, the assay can then be set up. The assay consist of the following:

Postive Control: 40ul of cells + 10ul of Ampicillin (10ug/ml)
Negative Control: 40ul of cells + 10ul of autoclaved water
Data points: 40ul of cells + 10ul of Ampicillin (at various concentrations)

The whole microtitre plate incubate overnight at 37 degrees celcius. The results are read the next day using the luminescence method. This is somewhat similar to what we doing in the laboratory using the spectrophotometer. The difference is that the spectrophotometer measures turbity, while in this case, the luminescence is measure. A dye called resazurin is added into the individual wells. Resazurin is an oxidation-reduction indicator which changes from non-fluorescent blue to fluorescent pink by oxidoreductase within living bacteria. Simply put, if there is the presence of S. aureus in a particular well, resazurin would change from blue to pink. If there is absence of S. aureus in a particular well, resazurin remains blue.

The luminescence produced by resazurin is read using a machine which measures the amount of luminescence produced. The data is then plotted into a graph, and the IC50 of Ampicillin can be determined. The IC50 of Ampicillin is the concetration of ampicillin which inhibits 50% of S. aureus, and is useful when used to compare the IC50 of the different types of tea.

Please feel free to ask me questions, I'll do my best to answer them as soon as possible. Thank you.

Regards,
Zhu Zhijie Dennis
0700847G

DxC 800 analyzer-continued

Hello! this entry is an addition to explain my previous entry on the DxC analyzer. Some of you may not understand how the analyzers in the lab use e principles to carry out the different kinds of tests. So, I have added in one example of test used by the DxC 800 Beckman Coulter analyzer used in the lab I'm attached to :)

Clinical Chem Lab technique
Name of test: Renal Function Test on DxC 800 analyzer
Principle of test: ISE (Ion Selective Electrode) Method

The DxC 800 analyzer can determine Sodium ion concentration by indirect potentiometry using two glass Sodium electrodes, whereby one of them will be used as a reference electrode. A reference electrode is the electrode with stable and well-known electrode potential. The outer layer of the glass electrode must be hydrated adequately. The sodium concentration is measured using a precise volume of sample that is mixed with a buffered solution. When the sample buffer mixture gets in contact with the electrode, sodium ions in the sample will undergo an ion exchange process with the sodium ions in the hydrated layer of the electrode. Thus, there will be a change in electrode potential which will be referenced with the reference electrode. From here, the concentration of Sodium can be measured.

Normal reference range of test results are:
0 day to 12 years old: 131-144mmol/L
> 12 years old: 135-145 mmol/L

Any results higher or lower than the normal reference ranges indicate abnormalities in patients.

This sodium measurements is used in the diagnosis and treatment of renal failure, hypernatraemia, hyponatraemia as well as other diseases involving electrolyte imbalance.

I hope this helps you guys to understand better! :)

Stella
0701059H
TG01

The Urine Bench

Good evening lecturers and fellow course mates. My name is Chu De Ming Jeremy (admin no. 0702919B) and I am attached to Microbiology (Bacteriology) for the whole course of attachment. I will be sharing my experiences over the past 2 weeks in my assigned laboratory. I shall try to keep my posts as clean and concise as possible to ease the monotony.

Before I begin, I apologize for this late post and to make up for this, I will try to share my encounters weekly. Today, I shall start of with my very first week (22nd June - 27th June) in the lab. I title it, The Urine Bench.

The Urine Bench

Urine culture is a very common laboratory test performed to diagnose urinary tract infections (UTIs) which are frequently due to bacterial infections. Escherichia coli, Klebsiella, Enterobacter, Proteus and Pseudomonas sp. are the common culprits of UTIs.
Urine culture requires midstream urine as the specimen because the first bit of urine that passes out may be contaminated from the skin. This is to eliminate possible normal flora growth which can interfere in our identification of pathogenic microorganisms.

In the lab, I was tasked to perform urine cultures for the whole week. Specimens arrived in batches, either in the form of urine, fluid from the kidneys, or dipslides which has already been dipped in urine.

Urine specimen is used to perform aerobic cultures on a split blood agar plate and cysteine lactose-electrolyte-deficient (CLED) media. Direct plating is performed using a disposable 1 micron wired loop and the plates are later sent for incubation at 35°C overnight.
Percutaneous nephrostomy (PCN) is performed on fluid from kidneys. Procedures are similar to aerobic cultures of urine. The only difference is direct plating require the use of both 1 and 10 micron wired loop on 2 separate split blood/CLED agar plates.



A self drawn picture illustrating how streaking is done. Urine is streaked onto blood agar first, then onto the CLED agar. This is to allow trace back if there is any errors or contaminants encountered.




When dipslides are received, they are sent for incubation at 35°C overnight.










Picture of dipslides from http://www.solarbiologicals.com/pages/medical/images/cled-mac-2.JPG
One side is CLED media, the other can be MacConkey (MAC) agar or blood agar.

Blood agar is an enriched differential media that enhances the growth of fastidious microorganism.
CLED media, due to its electrolyte deficient property, prevents Proteus sp. from swarming. It also contains lactose which differentiates lactose fermenters (yellow colonies) from non-lactose fermenters (blue colonies).
MAC agar is a selective differential media that inhibit the growth of most Gram-positive bacteria to stain Gram-negative bacteria for lactose fermentation.

Dipslides are unpopular compared to plating on agar plates as their area is small, making it difficult to make observations. However, one advantage of dipslides is that it can be retained for 2 days before sending it for incubation, as compared to urine where it has to arrive fresh (not more than 24h if kept in refrigerator).

Well, that is all you will need to know for The Urine Bench! Any questions please do not feel hesitant to ask. Thank you.
Check back for the next post in the next couple of days.

Jeremy.