Microbiology

Hello children!!!!

Haiz, so refreshing to go back to school for a change. Nonetheless, i must admit that i'm getting a little too used to the ole working life. Haha. I know some of you already are enjoying yourselves riiight.

So anyway, its been 10weeks (no way man!) and I've been posted to almost all the benches in my lab - urinalysis, microbiology, hematology, blood banking, order entry and clinical chemistry. Next month, i might even get a chance to follow the phlebotomists on their rounds to the wards and observe how they draw blood. Super cool. And and! Recently, Pei Shan (we're in the same lab) & I underwent a Point-of-Care Testing training as we will be sent out to do an Outreach Programme next month at a shopping mall. Ultra cool. So I guess that means, I can go back to school and apply my finger-pricking skills to my classmates. Mwahaha.

I'm sorry, I'm too used to digressing. So this week, i'm going to blog about a Stool Toxicological test using a testkit called ImmunoCard.

Name of Test: Detection of Clostridium difficile Toxin A & Toxin B in Stool

Introduction:
Clostridium difficile is a species of gram-positive rod-shaped bacteria that is the main cause in infectious diarrhea. Most strains of this bacteria produce 2 biologically and immunologically different toxins: Toxins A & B. A rapid test such as this ImmunoCard test enables the physician to verify any possible infection quickly and begin treatment immediately.

Principle:

• This is a rapid enzyme immunoassay
• Consists of a membrane held in a plastic frame with 2 sample ports and 2 reaction ports. The left-hand side column represents the Control panel while the other column represents the Test panel.
  

• This membrane contains immobolized antibodies to Toxins A & B. An Enzyme Conjugate, Wash Buffer, Substrate Reagent and Specimen Diluent is also provided along with the kit. The Enzyme conjugate contains antibodies to Toxins A&B coupled with horseradish peroxidase. The diluent is a buffered protein solution.
• Patient stool sample is diluted with the Specimen Diluent and the Enzyme Conjugate is added to it. It is then incubated for 5 mins. During this time, if there is toxin present, the molecules of the toxins will bind to the antibodies in the Enzyme Conjugate.
• Once incubation period is over, an aliquot of the sample is added onto the sample ports and incubated for another 5mins at RT. This time, the toxin-enzyme conjugate is separated from any particulate matter as it seeps through the membrane to the Test and Control reaction Ports.
• The toxin-conjugate complexes are captured in the Test port by immobolized anti-toxin. Then, the Wash Reagent, which is also a buffered similar to the Sample Diluent, is added to both ports. The reaction ports are then incubated again for 5 mins. At this point in time, the enzyme (HRP) modifies the Substrates Reagent, causing a colour change.
• The test kit is then read visually after incubation. The Control Port should be blue in colour while the Test Port with a blue colouration indicates presence of Toxins.

Test Results:
As this is not a quantitative test, there are no reference ranges. Test results will only indicate the presence of absence of Toxins A&B of C. difficile.

A positive result is indicated by a blue colouration in 2 Reaction Ports. A blue colouration in the Control Reaction Port (upper left) means that the sample has been added, reagents were active at the time of use and that proper sample migration has occurred.

If there is only blue colouration on the Control Reaction Port (upper left) and the Test Reaction Port (upper right) is colourless, then this would be a negative result.

Clinical interpretation:
As mentioned this test is to determine the presence of Toxins A&B of Clostridium difficile in human stool. It is part of a test called Stool Occult test. This test is used to aid physicians in diagnosing C. difficile-related diseases.

Alrighty, I'm done =). We've got 10 weeks more to goooooooo! Have fun all!

The cutest Lab Freak,
Sharifah

MCT techniques and a the machines stuff..

Hmm..okie..it's my turn again after Nat..this time I'm going to talk more about my MP.

Basically, my MP is about the protein analysis of lung cancer cells that are treated with herb using LC-MS/MS. It mainly involves MCT techniques and chemical engineering stuff such as the machines used. Prior to using the machines, I have to seed cells and here's where all the aseptic techniques and cell counting comes in. Using a hemocytometer, the cells are counted and the concentration of the cells are calculated. This had been elaborated in Najib's entry, so take a look if u guys don't know how to alright! A cell densitiy of 7million cells are seeded and 3 of the culture dishes are treated with 2mg/ml of herb extract after a 24 hr incubation time. Why 2mg/ml then? well, this concentration had been verified by a senior that it is the optimal concentration that will cause a lowest cell viability. An incubation time is required to allow the cells to adhere to the surface of the culture dish and also to proliferate somemore! After that, the cells are killed!! M-PER is added to allow the cell lysis so that protein extraction can be done using a cell scraper. The supernatant is then taken out and stored at -80degrees after a centrifuge step.

Now comes the part that is rather foreign to us, as it's rather chem eng based. 2D-LC is run! Before this, the proteins are trypsin digested and iTRAQ labeled. iTRAQ reagents will label proteins based on the different mass, namely 114,115,116,117. These reagents binds to the peptides (i.e. proteins after the trypsin digestion) at the N terminal of the lysine chain. This labeling will allow the quantification of the different proteins identified. In addition, it allows up to 4 samples to be mixed and run together, therefore lowering the time needed for the run to be carried out! This step then makes my life easier, because i will know if the protein is being up or down regulated based on the iTRAQ ratio that is given.

Ok! back to 2D-LC. 2D-LC is actually a fractionation step to fractionate the peptides samples. This involves 2 column modes, column 1 and column 2 mode and 2 different pumps are also involved, the quaternary pump and the capillary pump. In column 1 mode, the quat pump is the busy one, it pumps the sample that is injected into the Strong Cation Exchange (SCX) column then to the enrichment column and lastly to the waste. The peptides will then be stuck at the SCX column. In column 2 mode, the cap pump becomes the busy one. It pumps the peptides that is stuck at the enrichment column into the RP column. Erm, as some of the peptides will be still stuck at the SCX column, salt of different molarity is run. These salts help to elute the stuck peptides that is at the SCX column into the enrichment column. The irritating thing about this machine is that it lacks a auto fractionator, which means that I'm the fractionator. The eluted samples are collected at a 2mins interval. Which means that at every 2min, I have to change the tube to a new eppendorf tube. Imagine sitting at the lab for straight 4 hrs!! Can't leave the lab at all. Sickening rite!

Now here comes MALDI. MALDI helps in the protein detection and the protein will then be identified by the MASCOT system. I will not be elaborating on MALDI as Sharhirah had already talked about.. so guys..refer there! :)

Okie..thats about all..Just a brief idea on what my MP is actually about and the stuff involve.. If there's any questions, just post them k..I'll answer them asap!

Have Fun people! Back to school soon..haha..

Charmaine
0503186I

back to me agn (:

ouh. when i am blogging, it means that 2months of sip have passed!
how time flies eh?

Ha. Anyway back to serious business.
Yes today i shall post bout the experience i had and what i've learnt in the Mircobiology lab.
A short brief one that is. (=
Basically the microlab consists of 5 benches; namely,
1)Specimen Processing Bench; whereby all the specimens are being processed and sorted out.
2)Urine Bench; whereby necessary interpretation of primary urine culture plates(that are cultured the day before) and performing of tests for all positive tests.
3)Microscopy Bench; its whereby the staff in-charge process specimens such as sputum for diagnosis of TB/myobacteria infection.
4)Blood Bench; its the same process as in the urine bench, just that the specimen is of blood.
5)Miscellanous Bench; as its name called, its of interpretation and culturing of other specimens than blood and urine.

I shall focus more on the Bacteria ID Identification procedure. Its rather interesting, the best part i liked! and its bascially like the die-die-must-know thing in a mircolab. lol

First, gram-staining is done on the unknown bacteria.
Then by using the microscopy lens, identify the bact is of a gram positive (violet colour) or a gram-neg(pink colour).

For the gram-pos bacteria,
a Catalase test is done.
If its catalase positive- it is a Staphylococcus bacteria.
A Coagulate (latex) test is done to further identify its type.
So if its catalase pos, it is either a Stap.aureus or MRSA.
If its a catalase neg, it will be reported as STCH (normal commensal flora).
For catalase neg- it is a Streptococcal organism. Further tests e.g. alpha test, beta-grouping test etc are being carried out. Normally, the beta-grouping method is the most common, it tell us what group type it belongs to.

For gram-neg bacteria,
we'll carry out the biochemical tests (i.e. 7tubes of different tests + F12)
F12 is of antimicrobial agents such as penicillin, it tells us the sensitiveness and susceptibility of the bacteria towards different types of antibodies.
Biochemical Tests are of the followings:
1)KIA slant
2)Simmons citrate
3)Urease
4)Motility (& OF) test
5)Indole
6)Phenylalanine slant
7)Malonate test

So, by identifying the positive or negative results of the various tests will help us to differeniate which Enterobacteriaceae group it belongs to!

okay i am done with all the brief short explaination.
If you guys need futher explaination on the principles and the result interpretation of the various biochemical tests, just give me a message! i'll post it asap okay.

Have fun SIP-ing guys/babes. (:


signed,
Natalie

HOT MCT Techniques.

Elo Elo eh eh eh. Haha.

Hey guys wassap. Its been 7 cool/hot weeks since sip started. Its great working in the department of experimental surgery. Its cool looking at the animals but i am doing only invitro work, therefore i do not handle the animals.

In this post i will cover alot on mammalian cell technology stuff as this is the bulk of my experiments. Hope this will also freshen your memory.

Now lets got to the basics which is subculturing of cells. As charmaine mention earlier subculture is done when the cells have reached confluence. In my lab i will subculture the cancer lines (eg. Hep G2) when it is 80-90% confluent. Let my give 2 major importance of subculturing.

1) To prevent overcrowding of cells. Therefore porvides space for the cells to grow as over crowding will result in scenescence of the cells.

2)Subculturing replenish the nutrients contained in the media. Metabolic wastes which could be harmful to the cells at high concentrations are removed during subculturation.

If the cells have not reached confluence, we have to change their media about once or twice a week depending on the cell type. Certain cell lines are slow proliferators while others grows very fast. These slow proliferators will eventually use up all the nutrients and produce waste therefore a media change is needed for them.

Here are the steps to perform subculture. It is different from what we learnt during MCT lessons.

A) Subculturing of Cells (perform in Biosafety cabinet)
1. Discard spent media from culture flask into waste bottle
2. Wash with 10mL PBS and discard
3. Add 2mL of trypsin and incubate for 2- 3 minutes
4. Observe under the inverted microscope if cells are detached
5. After cells are detached, add 8mL of media to deactivate the trypsin and mix well
6. Transfer 10mL of cell suspension into a 50mL centrifuge tube
7. Centrifuge the cell suspension at 1500 rpm for 3 minutes
8. Discard supernatant and resuspend cell pallet while washing with 10mL PBS
9. Centrifuge at 1500 rpm for 3 minutes
10. Discard supernatant
11. For a cell concentration ratio of 1:20, add 10mL of media and resuspend the pallet
12. Prepare a new culture flask adding 19.5mL media
13. Aliquot 0.5mL of cells into new 75cm2 culture flask containing media
14. Check under the inverted microscope for cells
15. Incubate cells in the 370C, 5% CO2 incubator

The difference is in Step no. 6 onwards.

In my lab, after deactivating the trypsinized cells, there is one additional stet which is the washing with PBS (step no. 8). This is done so as to remove as much trypsin as possible. That is why in our MCT lab sessions, some of us had a hard time detaching the cells with trypsin as previously, the trypsin is not fully removed by an additional washing step. The cells are adapting to the enzyme trypsin effect and thus it takes a longer time for the trypsin to work for subsequent trypsinization. Also in my lab, culture flask are changed every time subculturing is done. I guess our school do not do such as it is too costly and it is not for something of high importance such as research. Changing flask regularly helps prevents contamination as older flasks are exposed to the environment which contains many microbes.

Now let me talk about our favourite hemocytometer. Guess what it is used for? Anyone?

Correct! It is used for cell counting. For mammalian cells, cell counting is done for many reasons. Examples are when freezing cells, performing MTT assay and many more. For me i had to perform cell counting as I need to do MTT assay.

For cell counting, I obtain the cell number estimate by counting big(1mm by 1mm) squares at the 4 corners of the counting chamber.

*Sorry ppl im unable to put a nice/hot photoshoot of the counting chamber cos blogger is being a pain for now.*

Trypan blue is used in cell counting to differentiate the dead cells from the living ones. The dead cells will be stained blue whereas viable cells will look white under the microcope. Formula for cell concentration:

C=n x 10^4 x df

Whereby,

c=cell concentration (cells/ml)

n=average no. of cells/mm^2 (average of the 4 squares)

10^4=volume counted

df=dilution factor

Cell counting is crucial in MTT assay as seeding of cells in each of the wells of the 96-well plate must contain equal amounts of cells so that accurate results will be produced when the assay is performed.

Ok guys my post ends here and pls do ask any questions if you are in doubt. Have fun and hope you guys learn alot during SIP. Take care.

Najib Bin Hamid (0503217B)
TG01

Hi everyone! Sorry for the delay in my post there was a bit mixed up in my remembering of the weeks.

I am working in the food and water microbiology lab now. In the lab, the main testing that we were instructed to focus on is on the testing of food samples. Ying Ying has roughly described of routine testing of food and the basics of food tesing principles. Now I am going to touch on the testing of specific pathogen, Salmonella. The most common testing for Salmonella in our lab is the testing of chocolate samples from overseas companies.

For Salmonella testing of chocolate sample:

25 grams of chocolate sample is being weighed and 225ml of skim milk is being added to the chocolate sample. For normal Salmonella testing, buffered peptone broth is being added instead of skim milk. However, as this is a request from the company overseas, we have to follow their instruction as that is their way of testing there. After the addition of skim milk, the food is being homogenized by the stomacher. The stomacher has been described in Ying Ying’s blog posting so I shall not talk about it anymore. After stomaching at 230rpm for 30s, phosphate buffer is then added to the homogenate. After that, the chocolate homogenate is then taken to the incubator for incubation at 37◦C.

After incubation for 24 hours, 1ml of the chocolate homogenate is being pipetted into Tetrathionate Brilliant-green Bile Enrichment Broth (TTB) and 0.1ml is pipetted into Rappaport and Vassiliadis (RV Broth). This is a selective enrichment step to improve the growth of Salmonella. For TTB, 0.2ml of iodine and 0.01ml of brilliant green is added before the chocolate sample is added.

For TTB (incubate at 35◦C):
- Only organisms which possess the enzyme tetrathionate reductase can grow (Only Proteus and Salmonella)
- Proteus can be inhibited by adjusting the pH to about 6.5 or the addition of novobiocin.
- Bile salts promote the growth of enteric bacteria and inhibit the growth of bacteria that do not normally live in the intestine.
- Brilliant green inhibits gram-positive bacterial flora.
For RV Broth (incubate at 42◦C):
- Allows only Salmonella growth by exploiting the following characteristics of Salmonella:
o Ability to survive at high osmostic pressure
o Multiply at low pH values (pH: 5.5)
o More resistant to malachite green (increase amounts)
o Less demanding nutritional requirements

After incubation for 1 day at their respective incubators, the samples from both the TTB and RV Broth is streaked onto Hektoen Enteric (HE agar), Bismuth Sulfite (BS agar) and Xylose Lysine Desoxycholate (XLD agar). 3 different types of agar plates are used as each of them supports the growth of different Salmonella species so any form of Salmonella can be detected.

Mode of action:
Hektoen Enteric agar
- Contains bromothymol blue and acidic fuchsin indicators. Differentiates lactose-positive colonies from lactose-negative colonies.
- Additional carbohydrates (sucrose and salicin) helps early detection of slow lactose-fermenting organisms to prevent false-postive results.
- Thiosulphate and ferric citrate causes H2S positive colonies to become black in colour.
- Blue green colonies with black centres would indicate presence of Salmonella..

Bismuth Sulfite agar
- Selective media for isolation of salmonella species.
- Bismuth Sulfite and Brilliant Green act together as a selective agent by suppressing the growth of coliforms.
- H2S production by salmonella due to presence of sulphur.
- Reduction of bismuth ions forms metallic lusture around the colonies.
- Black centre, light edges surrounded by a black precipitate with metallic luster would indicate presence of Salmonella.

Xylose Lysine Desoxycholate agar
- Degradation of xylose, lactose and sucrose to acid causes phenol red indicator to change to yellow.
- Decarboxylation of lysine by salmonella species alters pH to alkaline causes purple colouration around the colonies.
- H2S production indicated by thiosulfate and iron(III) salt forms a precipitate of black iron sulfide in the colonies.
- Red colonies, translucent, sometimes with a black centre would indicate presence of Salmonella.


If there are any suspicious colonies, biochemical tests should be carried out.
The following descriptions below are the biochemical tests for the confirmation of Salmonella.

Triple Sugar Iron (TSI) differentiates gram-negative enteric bacilli based on carbohydrate fermentation and production of hydrogen sulphide. TSI contains dextrose, sucrose, lactose, phenol red (detection of carbohydrate fermentation) and ferrous ammounium sulphate (detection of hydrogen sulphide). The gas produced during carbohydrate fermentation will change the phenol red indicator from red to yellow. Formation of hydrogen sulphide will cause blackening in the butt of the test tube. To facilitate the detection of organism that only ferments dextrose/glucose, the dextrose/glucose concentration is 1/10 the concentration of lactose or sucrose. The acid produced at the slant oxidizes rapidly, causing the medium to remain red or revert to an alkaline pH. However, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. Loosely capping will allow the exchange of gases.

Lysine Iron Agar (LIA) aids the differentiation of enteric bacilli based on the ability to decarboxylase lysine and to produce hydrogen sulphide. LIA contains bromcresol purple indicator (detection of lysine decarboxylase production), lysine (detection of enzymes lysine decarboxylase and lysine deaminase), ferric ammonium citrate and sodium thiosulfate (both are indicators of hydrogen sulphide). The gas produced during carbohydrate fermentation will cause the bromcresol purple indicator to remain purple. Formation of hydrogen sulphide will cause blackening of the medium due to production of ferrous sulfides. Production of lysine decarboxylase would cause an alkaline reaction (purple). Deamination of lysine would cause an acid slant (red).

Urease Broth is used in parallel with TSI and LIA. Used for the detection of Proteus organism as it causes hydrolysis of urea (colour change to pink).

Indole test is used in the differentiation of genera and species. Tryptone water is used due to its high content of tryptophan. Any microorganism with the enzyme Tryptophanase will cleave tryptophan to produce indole and other products. Kovac’s reagent will then react with indole to form a red ring. False negative results will be produced if the pH falls below (7.4-7.8).

Methyl Red test is used to test the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Organisms that form large amounts of acid from glucose fermentation causes the pH to fall below 4.4 (colour becomes red).

Voges Proskauer (VP) test is used to detect the production of acetylmethylcarbinol (acetoin) from the fermentation of glucose/dextrose. If positive, acetoin is oxidized in the presence of oxygen and potassium hydroxide to diacetyl which reacts with peptone water to give off a red colour.

Decarboxylase media are used in differentiation of gram-negative enteric bacilli based on the production of arginine dihydrolase and lysine and ornithine decarboxylase. When dextrose is being fermented, the bromocresol purple indicator changes from purple to yellow. The acidic condition also stimulates decarboxylase activity. When the amino acid is degraded, a corresponding amine is being produced. The amine then elevates the pH of the medium, causing a colour change of the indicator from yellow to purple.

Simmon’s Citrate agar is used for the differentiation of gram-negative enteric bacteria on the basis of critrate utilization. Organisms that are able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole source of nitrogen and carbon will produce an alkaline reaction by changing the bromthymol blue indicator from green to blue (alkaline).


After incubation, if the biochemical tests results are as follow, the chocolate sample can be presumed to contain Salmonella:

TSI (incubate at 35◦C for 24hours): Acid butt (yellow), alkaline slant (red) with H2S production (black)
LIA (incubate at 35◦C for 24hours): Alkaline reaction (purple) with H2S production (black)
Urease test: Negative (no colour change)
Indole test (Tryptone water + Kovac’s reagent): Negative
Methyl Red test: Positive (distinct red colour)
VP test: Negative (no colour change)
Base decarboxylase: Negative (yellow)
Glucose/Xylose decarboxylase: Negative (yellow)
Lysine decarbosylase: Positive (purple)
Ornithine decarboxylase: Positive (purple)
Simmon’s Citrate: Blue
Gram staining: Gram-negative bacilli

After that, serological test is being carried out. Each presumptive Salmonella isolate (using TSI agar slant capture) is being tested against Polyvalent O (somatic) and Polyvalent H (flagella, phase I and II) antisera. For both Polyvalents, there is agglutination. This shows that the presumptive Salmonella can be confirmed as Salmonella.

Jeremy Lee
TG01
0503168G