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.
Tuesday, July 28, 2009
Sunday, July 19, 2009
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
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
Sunday, July 12, 2009
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
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
Tuesday, July 7, 2009
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.
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.
Saturday, July 4, 2009
Week 1 - DxC 800 analyzer
Hi people!
Stella here.
i'm currently attached to Biochemistry Lab.
as it is an automated lab, we get to see different types of machines and so i'll be mostly talking about analyzers.
The first week of work, i was assigned to the Beckman Coulter DxC 800 Analyzer.
Its function is to run different types of tests such as liver function test, renal function test and cardiac markers.
DxC 800 makes use of three principles in determining the sample results.
They are Enzymatic Method, ISE(Ion Selective Electrodes) Method and Spectrophotometry methods.
The analyzer contains different types of reagents, serving different purposes of the biochemical analysis. It is divided into two parts. They are cartridge chemistry which contains reagents such as ALT, AST, CK and many more. Whereas the modular chemistry, it contains sodium, potassium, chloride, glucose and many more.
Cartridge chemistry makes use of spectrophotometry methods whereby a substance will be produced and is able to absorb light at certain wavelengths. This happens when a sample is mixed together with the appropriate reagents.
As for modular chemistry, the ISE will convert the activity of a specific ion which will be dissoved in a solution into an electricity potential. The electrode dat senses is usually made up of an ion-specific membrane, alongside with a refernce electrode.
It is important to top up the reagents at different intervals during the day to make sure that reagents do not run out and patient samples can assessed without any disturbances.
Each time the reagents need to be topped up, the system will prompt the person on duty.
I was given the chance to top up the reagents, as well as loading the patient samples manually into the analyzer.
I also learnt all about calibration and quality control which have to be done every day, preferably in the morning.
As the lab is mostly automated, the analyzer plays an important role in producing results for the patient samples so that diagnosis and treatment of the diseases can be confirmed.
:)
Stella here.
i'm currently attached to Biochemistry Lab.
as it is an automated lab, we get to see different types of machines and so i'll be mostly talking about analyzers.
The first week of work, i was assigned to the Beckman Coulter DxC 800 Analyzer.
Its function is to run different types of tests such as liver function test, renal function test and cardiac markers.
DxC 800 makes use of three principles in determining the sample results.
They are Enzymatic Method, ISE(Ion Selective Electrodes) Method and Spectrophotometry methods.
The analyzer contains different types of reagents, serving different purposes of the biochemical analysis. It is divided into two parts. They are cartridge chemistry which contains reagents such as ALT, AST, CK and many more. Whereas the modular chemistry, it contains sodium, potassium, chloride, glucose and many more.
Cartridge chemistry makes use of spectrophotometry methods whereby a substance will be produced and is able to absorb light at certain wavelengths. This happens when a sample is mixed together with the appropriate reagents.
As for modular chemistry, the ISE will convert the activity of a specific ion which will be dissoved in a solution into an electricity potential. The electrode dat senses is usually made up of an ion-specific membrane, alongside with a refernce electrode.
It is important to top up the reagents at different intervals during the day to make sure that reagents do not run out and patient samples can assessed without any disturbances.
Each time the reagents need to be topped up, the system will prompt the person on duty.
I was given the chance to top up the reagents, as well as loading the patient samples manually into the analyzer.
I also learnt all about calibration and quality control which have to be done every day, preferably in the morning.
As the lab is mostly automated, the analyzer plays an important role in producing results for the patient samples so that diagnosis and treatment of the diseases can be confirmed.
:)
Thursday, July 2, 2009
Week 1
First of all, we would like to apologize to Ms Chew and our fellow coursemates who were anticipating for our first post in Week 1 of SIP. Our group will be doing 2 posts this week to compensate.
Hi this is Hakim from TG01.
Since this is our first post, I think it would be best if I talk about the beginning of the laboratory procedures, which is the specimen reception in the main branch. I was posted to Specimen Reception for a day, but I learnt a lot of things. Specimen reception is one of the most important phase as the samples have to be checked and verified before being sent for testing. Samples are brought in in batches from different areas. Samples are usually brought in by couriers and usually they go and collect samples from different clinics more than once a day.
Specimens come with forms indicating the name and the type of test needed to be done (e.g. culturing of swab or HIV test on blood). The type of specimens received must also be correlated with the forms to make sure that there is no missing or lost items. Usually, there are red coloured forms which indicate that it is "Urgent". Therefore, they are of higher priority and results have to be released ASAP. All samples received early in the morning (i.e. 9 am) also are indicated as "Urgent".
After that, barcodes are placed on the form as well as the specimens (e.g. tube, bottle, swab container) so that the specimens can be registered in the database (LIS). I'm familiar with the procedures with the registering of tests and patient information for the TMC branch, but I'm not too familiar with the procedure in the main lab.
All blood samples (excluding those with anticoagulants such as EDTA) are to be centrifuged to seperate the serum from the red blood cells.
The forms are placed in a pidgeon hole so that the different sections are able to perform the tests.
Hakim
0703555C
Hi this is Hakim from TG01.
Since this is our first post, I think it would be best if I talk about the beginning of the laboratory procedures, which is the specimen reception in the main branch. I was posted to Specimen Reception for a day, but I learnt a lot of things. Specimen reception is one of the most important phase as the samples have to be checked and verified before being sent for testing. Samples are brought in in batches from different areas. Samples are usually brought in by couriers and usually they go and collect samples from different clinics more than once a day.
Specimens come with forms indicating the name and the type of test needed to be done (e.g. culturing of swab or HIV test on blood). The type of specimens received must also be correlated with the forms to make sure that there is no missing or lost items. Usually, there are red coloured forms which indicate that it is "Urgent". Therefore, they are of higher priority and results have to be released ASAP. All samples received early in the morning (i.e. 9 am) also are indicated as "Urgent".
After that, barcodes are placed on the form as well as the specimens (e.g. tube, bottle, swab container) so that the specimens can be registered in the database (LIS). I'm familiar with the procedures with the registering of tests and patient information for the TMC branch, but I'm not too familiar with the procedure in the main lab.
All blood samples (excluding those with anticoagulants such as EDTA) are to be centrifuged to seperate the serum from the red blood cells.
The forms are placed in a pidgeon hole so that the different sections are able to perform the tests.
Hakim
0703555C
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