Week 10

Hi everyone, it’s my turn again this week. For this week, I think I’m going to share about what I’ve learned from the microbiology section from my lab. I’m going to introduce this kit that can help to identify the different types of gram-negative bacteria. It is named the Microbact Gram-Negative Identification System.

Principle:
The Microbact Gram-negative system is a standardized micro-substrate system designed to stimulate conventional biochemical substrates used for identification of Enterobacteriaceae and common miscellaneous Gram-negative bacilli. Organism identification is based on pH change and substrate utilizations.

This product consists of two separate substrate strips, 12A and 12B. Each strip consists of twelve different biochemical substrate. The 12A strip can be used alone to identify oxidase-negative, nitrate positive glucose fermenters and can be used for screening pathogenic Enterobacteriaceae from enteric and urine specimens or identification of other common isolates. The 12B strip can be used together with the 12A strip for identification of oxidase positive, nitrate-negative, glucose non-fermenters and the Enterobacteriaceae.

Each kit contains one holding tray for the 12A and 12B strips and organism ID report forms including colour interpretation chart.

Examples of species of oxidase negative, Gram-negative bacilli that can be identified using 12A strip alone:
Acinetobacter spp., Shigella spp., Enterobacter spp., Escherichia spp., Salmonella spp., etc.

Examples of species of Gram-positive bacteria that can be identified by using combination of 12A and 12B strips:
Vibrio spp., Pseudomonas spp., Pasteurella., etc.

Set up procedure:
1.Isolation

To use the kit for identification of organism, a pure culture of that particular organism must be obtained. Appropriate agar media such as MacConkey, Blood or Chocolate may be used to grow the organism for approximately 18 to 24 hour. I believed you all would know how to do a pure culture plate by now so I won’t state the procedures here.

Before using the kit, an Oxidase test should be performed on that organism to be identified. As mentioned above, if it’s positive, 12A and 12B strips should be used and if it’s negative, 12A strips can be used alone.

2.Preparation of inoculum

Pick 1-3 isolated pure colonies from the 18-24hr culture and emulsify in 2.5 to 5 ml of sterile saline. Usually 1 colony is enough to emulsify in 2.5ml of saline if doing on the 12A strip alone. If it’s doing 12A and 12B strips, it may need 2 colonies to emulsify in 5ml of the saline. Then, mix thoroughly for a uniform suspension.

3.Inoculation

As the strips are sealed, the individual substrate sets can be exposed by cutting the sealing strip and slowly peel it back. Then, place the strip on the holding tray and using a sterile dropper, fill the bacterial suspension to half of each well in the set. After that, drop one drop of sterile mineral oil on the wells that have the substrates underlined (for 12A strip is well 1,2 and 3 while for 12B strip is well 8 and 12).


In case you all want to ask me what each well contains, here’s the answer:
For 12A strips-Well 1- Lysine
Well 2- Ornithine
Well 3- H2S
Well 4- Glucose
Well 5- Mannitol
Well 6- Xylose
Well 7- ONPG
Well 8- Indole
Well 9- Urease
Well 10- VP
Well 11- Citrate
Well 12- TDA

For 12B strips-Well 1- Gelatin
Well 2- Malonate
Well 3- Inositol
Well 4- Sorbitol
Well 5- Rhamnose
Well 6- Sucrose
Well 7- Lactose
Well 8- Arabinose
Well 9- Adonitol
Well 10- Raffinose
Well 11- Salicin
Well 12- Arginine

4.Incubation

Reseal the inoculated rows with adhesive seal and write the specimen identification no.(in my lab case is using barcode no.) on the seal with a marker. Then, incubate 33-37°C for 18 to 24hrs.

5.Reading the test strips

Remove the test strips from the incubator and unseal them for reading. Positive and negative reaction results are recorded down on the organism ID report forms by comparing the colour change with the colour chart. For some of the wells, there’s an additional reagent to add to before reading. For 12A strips, well 8 drop 2 drops of Indole (Kovacs) reagent and read within 2 minutes, well 10 add 1 drop of VPI and 1 drop of VPII and read within 15 to 30 minutes and well 12 add 1 drop of TDA reagent and read immediately. These additional reagents are required for the reaction to start as different bacteria can have different reaction to these reagents.

There’s also an additional test called the Nitrate Reduction Test. It is performed in well 7 (ONPG) after reading the ONPG reaction. 1 drop of Nitrate reagent A and 1 drop of Nitrate reagent B is added to the well and a change to colour red within few minutes indicates nitrate reduction to nitrite. The positive reaction indicates that it is an organism belonging to the family of Enterobacteriaceae.

6.Interpretation

For the interpretation is pretty easy as they adopted a coding system/program that interpret the type of bacteria simple by entering the recorded result. For every 3 reaction (every three wells), it is added up to a single digit of the code. So if only 12A strip is used, there will be 4 digits which make up the code. If using 12A and 12B strips together, there will be a total of 8 digits that make up the code. Simply just choose the correct program and enter the code and the system will tell you what kind of bacteria is it.


Alright, that's it for this week. Enjoy your life people, if you still can now. Lol.

Sharon
Tg01

Week 9

Hi all, here I am again..

For my MP, I have started on doing my 'control' assay on the 96-well microtitre plate. This experiment is to determine the concentration of cells to be use in future experiments.

Yellow section(Row A-D): Positive control
Red section(Row E & F): Negative control(blank)
Blue section(Row G): Negative control(with 100ug/ml of ampicillin)
Green section(Row H): Negative control(with 200ug/ml of ampicillin)

Methods:
1. After taking the OD reading(till the bacteria cells reached the log phase), dilute the cells to get
the concentration of cells needed.
2. Using a multichannel pipette and micropipette,
For Row A-F, add 10ul of autoclave water into each well.
For Row G, add 10 ul of 100ug/ml of ampicillin into each well.
For Row H, add 10ul of 200ug/ml of ampicillin into each well.
For Row A-D and Row G &H, column 1-3, 4-6, 7-9, 10-12, add different concentration of
bacteria cells into the well.
For Row E & F, add fresh TSB as this will be the blank control of the assay
3. After adding all the solution into the wells, incubate the microtitre plate at 37oC overnight.
4. Next day, prepare Resazurin which is a redox active dye.
5, Add 10ul of Resazurin dye into all the wells and incubate it.
6. At 1 hour interval, take the fluorescence reading.
7. After 5 hours, all datas collected are to be sort and plot graphs using those data.
8. From the graph, determine which concentration of cells can give a better sensitivity.
9. In future experiment, the specific concentration of cells will be use.

In the real experiment, the 10ul of autoclave water will be replace by tea extracts.

As for my SIP, other then data entry, I also help to prepare acidulants by measuring 0.6g of citric acid, lime juice, vinegar and tartaric acid, and add 2L of DI water into it, then mix them. Other then helping to prepare media, I also help to check some items in the lab making sure all items are in place. From doing this, I realised how tiring the work of the TSO can be, so make sure the next time you use the lab, put all items back to their original place if not TSO will have a hard time looking for them.

Justina
0605950E
TG01

Department: Clinical Biochemistry Laboratory

During the fourth week of my attachment, I was stationed at the DxI station where I observed and learnt about the Beckman DxI-600 and its day to day maintenance.

Every morning, the technician-in-charge of DxI would be responsible for its daily maintenance such as

Backing up the system and data – By doing so, the data (Eg. The type of test run on which specific specimen and its results) accumulated in the previous day would be saved into floppy disks and could be used for further reference.)

Checking Inventory – This is important as it ensures that all reagents required by the DxI to perform all its tests are topped up and sufficient to last the entire day.

Recording Test count – This simply requires the technician to record the total number of tests performed by the DxI the day before.

Shaking the solid waste container – The waste container is where DxI deposit solid waste such as pipette tips. Usually, the waste would accumulate at one side of the box (as the tips are dropped from the same spot) and shaking the box would balance out the level of the contents.

Running a daily special clean – This daily special clean is commercially prepared by the DxI manufacturer. The special clean solution is transferred to a reaction vessel (plastic holding cups) by a dropper and the reaction vessel is then placed on a specific rack that carries the solution into the DxI once the task is assigned for the rack to run. This daily special clean is used for cleaning probes in the DxI. Probes are in fact mechanical arms that the machine used to pipette and transfer specimens and reagents within the DxI. Although it is constantly washed by the machine, running a special clean very morning would ensure removal of possible specimen/reagent traces from the probe itself and its pipes.

Other than its daily maintenance, I have also researched on the one of the common tests carried out by the DxI – Insulin.

Brief overview of Insulin:
Insulin is a hormone secreted by the pancreas that facilitates the uptake of glucose from the blood and storing it as glycogen in the liver and muscle. Glycogen (fuel storage) would be broken down to glucose when energy is required by the body later on.

When insulin level is low or absent, glucose is not taken up from the blood and stored the liver or muscle cells. The glycogen storage would eventually deplete as the body utilizes energy, this would then results in the body utilizing body fats as energy source. At the same time, glucose in the blood remains high as insulin fails to function. This condition is known as diabetes mellitus.

Test Principle:
DxI is able to test the levels of insulin in patient’s serum by using the ultrasensitive insulin assay which is an immunoenzymatic sandwich assay.

1. A sample is added to a reaction vessel with monoclonal anti-insulin antibody and anti- insulin alkaline phosphatase conjugate.

2. Insulin in the patient’s serum will bind to the anti-insulin antibody on solid phase, while the conjugate reacts with different antigenic sites on the insulin molecule.

3. Unbound materials are washed away.

4. The chemiluminescent substrate LumiPhos 530 is added to the reaction vessel and light generated when this substrate react with the conjugate is measured with luminometer.

Additional notes:
Patient’s serum are used for insulin testing and as red blood cell contains insulin-degrading enzymes, badly hemolyzed samples are not suitable as insulin levels may be suppressed, causing pre-analytical variations and inaccurate results.

Tan Zhao Rong
Tg01

Special Stains

Subject: Histopathology

Hi guys!
This is my 7th week at Histopathology, and so we have gotten pretty familiar at how things are managed and operated there. My previous post mentioned a bit on Special Stains, so now I'm here to share about 2 of the more common ones.

*Note:

• Commerical reagents are usually used in the lab
  
• Timings in the staining procedure are for your reference only because I've learnt that different types and different sections of tissues absorb and expel dye molecules at different rate.
  So if you are staining 5 slides at the same time using the same timing, the results obtained for each slide will vary. Thus, it is important to perform microscopic examination for each slide after each staining.
  However, there are also some stains which can be left on the sections as long as you want as the duration does not affect the staining.
  
• All reagents have their specific "lifespan" so it is important to record the date when the bottle of reagent is opened and their expiry date. Reagents are changed as often as needed to ensure quality staining.
  
• All types of staining including H&E are to be done with a control (positive control) section. It can either be at the upper half of the same slide, or on another new slide. This is added for the pathologist as a reference during microscopic examination.

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Alcian Blue (ALBL)

Function:
· Stains acid mucins and acid mucosubstances

Where can acid mucins and acidic mucosubstances be found?
· Excessive amount of non-sulphated acidic mucosubstances can be seen in mesothelioma, with certain
amount occurring normally in blood vessel walls but increased amount in early lesion of atherosclerosis.

Principle:
· Alcian Blue is a group of polyvalent basic dyes that are water soluble
· Blue colouration is present due to presence of copper in the dye molecule
· Alcian Blue at pH 2.5 stains both sulphated and carboxylated sialomycins (gylcoproteins)
· Alcian Blue forms self-linkages with acidic groups of acid mucopolysaccharides

Control used:
· Small intestine or colon

Fixative used:
· 10% Neutral Buffered Formalin

Chemicals required:
· 1% Alcian Blue
· 3% aqueous Glacial Acetic Acid
· Nuclear Fast Red

Reagent preparation:

1. Alcian Blue
· Alcian blue – 1g
· 3% aqueous Glacial Acetic Acid – 100ml

Allow the dye to dissolve and adjust the pH to 2.4 by using 1 M of sodium hydroxide. Store in a dark bottle to prevent reaction of dye with the light. This reagent is stable at room temperature for 6 months.

2. 3% Glacial Acetic Acid
· Glacial Acetic Acid – 6ml
· Type II water (DI Water) – 200ml

Store at room temperature.This reagent is stable for 1 year.

Staining procedures:
1. Heat the slide containing the section on the hotplate for 3 minutes
2. Dewax the section using the auto-stainer (machine)
3. Bring section to water to prevent section from drying up
4. Rinse the section using 3% aqueous Glacial Acidic Acid solution
5. Stain in 1% Alcian Blue in 3% aqueous Glacial Acidic Acid for 20 minutes
6. Rinse in 3% aqueous Glacial Acidic Acid solution before rinsing in water
7. Counterstain with Nuclear Fast Red for 5 minutes
8. Rinse the section with water
9. Dehydrate the section using graded alcohol from 95% to absolute alcohol
10. Clear the section using 3 changes of xylene
11. Mount the slide with Depex

Results:
· Acid mucins – Blue
· Nuclei – Reddish pink

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What is mesothelioma?

· Cancer of mesothelium
· Mesothelium is a thin membrane that surrounds most of our internal organs for protection purpose. The
membrane consists of 2 layers; one on the organ, another forms a sac around it. In the sac, there is a fluid
produced by the mesothelium to reduce friction as our organs glide against one another or against our
body structure such as the rib cage.

What is atherosclerosis?

· Hardening or arteries
· This is due to the buildup of deposits on the walls of the arteries. Common deposits are fatty substances,
cholesterol, calcium and cellular waste products.
· Buildup of deposits is known as plague and can rupture the arteries, causing the blood clots to obstruct
blood flow or travel to other parts of the body, resulting in heart attack, stroke etc.

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Masson Trichome (MT)

Function:
· Stains muscle, fibrin and collagen

Principle:
· A section is first treated with Masson Ponceau Fuchsin (small molecule anionic dye) before treated with
phosphomolybdic acid or phosphotungistic acid solution
· Phosphomolybdic acid or phosphotungistic acid solution will compete woth the dye to gain acess to the
collagen, at the same time, expelling the dye in the process
· The collagen is then treated with a larger molecule green or blue dye

Control used:
· Liver tissue, skin or muscle

Fixative used:
· 10% Neutral Buffered Formalin

Chemicals required:
· Haemotoxylin
· 4% aqueous phosphomolybdic acid
· Masson’s Light Green
· Ponceau 2 R
· Acid Fuchsin
· Glacial Acetic Acid

Reagent preparation:

1. Masson Ponceau Fuchsin
· Ponceau 2 R – 3.5g
· Acid Fuchsin – 1.75g
· Glacial Acetic Acid – 5ml
· Type II water (DI Water) –500ml

This reagent is stable at room temperature for 6 months.

2. 2% Phosphomolybdic- tungstic Acid
· Phosphomolybdic Acid – 4g
· Phosphotungistic Acid – 4g
· Type II water (DI Water) – 200ml

Dissolve in water and heat it at 50-60oC overnight by putting it in the oven.
Filter and store at room temperature.

3. 1% Acetic Acid
· Glacial Acetic Acid – 2ml
· Type II water (DI Water) – 98ml
Mix before use. Stable at room temperature for 6 months.

Staining procedures:
1. Heat the slide containing the section on the hotplate for 3 minutes
2. Dewax the section using the auto-stainer (machine)
3. Bring section to water to prevent section from drying up
4. Post fix the section in Bouin’s solution for 30 minutes at 56oC
5. Stain nuclei with Weigert’s iron haemotoxylin for 10 minutes
6. Wash in tap water and blue for 5 minutes
7. Stain in Masson’d Ponceau-Fuchsin for 5 minutes
8. Wash in 1% Acetic Acid
9. Stain in Masson’s Light Green for 30 seconds to 2 minutes, until collagen is green. In order to
control intensity of the stain, it is better to stain for 1 minute first, then rinse in acid water before examining
under the microscope.
10. Rinse in 1% Acid Alcohol
11. Dehydrate the section using graded alcohol from 95% to absolute alcohol
12. Clear the section using 3 changes of xylene
13. Mount the slide with Depex

Results:
· Nuclei – Blue black
· Cytoplasm – Light red
· Muscle – Dark red
· Red cells – Bright red
· Hyaline & fibrin – Bright red
· Collagen & mucin – Green

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Cheers!

Ting Ying Chee
TG01