Sunday, December 9, 2007

Group Post: MMIC dPBL-1

Biochemical and Culture Testing


CASE 1

Possible Organisms Biochemical Tests & Results Culture agars
Escherichia coli
Gram Stain: -
Oxidase: -
Lactose: +
Lysine: +
TSI slant: +
Indole: +
Methyl red: +
VP: -
Citrate: -

5% Sheep Blood Agar
Eosin-Methylthionine Blue Agar
MacConkey Agar
Pseudomonas aeruginosa
Gram stain: -
Oxidase: +
Pyocyanin: +
Fluoreseein: +
Citrate: +
Nitrase: +
Lipase: +
Ureases: +/-
Indole: -
TSI: -

Pseudomonas Agar P
Pseudomonas Agar F
MacConkey agar plate
Eosin-Methylthionine Blue agar
Cetrimide agar
Staphylococcus aureus
Gram Stain: +
Catalase: +
Coagulase: +

5% Sheep Blood Agar
Mannitol Salt Agar
Staphylococcus saprophyicus
Gram Stain: +
Catalase: +
Coagulase: -
Oxidase: -
Phosphatase: -
Urease: +
Lipase: +
2-hour PYR broth hydrolysis: -
D-trehalose; +

5% Sheep Blood Agar
Mueller-Hinton Agar Lawn with Urine
Enterococcus faecalis
Gram Stain: +
Catalase: -

5% Sheep Blood Agar
Chocolate Agar
6.5% NaCl Concentration Agar (halotolerant)
Bile Esculin Agar (BEA) Slants
Proteus-Providencia-Morganella
e.g. Proteus mirabilis

Gram Stain: -
Lactose: -
Oxidase: -
Urease: +
Indole: -
Nitrogen Reductase: +
Methyl Red: +
Vogues-Proskauer: -
Catalase: +
Phenylalanine Deaminase: +

MacConkey Agar
CLED
Klebsiella-Enterobacter-Serratia
e.g. Klebsiella oxytoca

Gram Stain: -
Oxidase: -
Lactose: -
Urease: -
Nitrate: +
Indole: +
Sucrose: +
Dextrose: -
Methyl Red: -
Vogues-Proskauer: +
Gelatin: -
Amylase: +
Lipase: -
Catalase: +
Citrate: +
H2S: -

5% Sheep Blood Agar
MacConkey Agar
Trypticase Soy Agar
Klebsiella-Enterobacter-Serratia
e.g. Klesiella pneumoniae

Indole: -
Sucrose: +
Dextrose: -
Methyl Red: -
Vogues-Proskauer: +
H2S: -
Lysine Decarboxylase: +

5% Sheep Blood Agar
MacConkey Agar

Done by Lizzie


CASE 2

Campylobacter jejuni
Oxidase (+)
Sensitivity to nalidixic acid

Blood agar with antibiotics to other fecal microbes
Fail to grow at 25C, must grow at 42C
Microaerophilic (grow at 5% oxygen + 10% carbon dioxide)
Shigella species
TSI (alkaline slant and acidic butt, with no gas and no H2S)

MacConkey's or EMB agar (colourless colonies)
Anaerobe
Escherichia coli
TSI (acidic slant and acidic butt, with gas ,but no H2S)
Indole test (+)

MacConkey's (pink colonies) or EMB agar (green sheen colonies)
Anaerobe
Clostridium perfringens
ELISA to detect Clostridium perfringens Enterotoxin

TSC agar containing egg yolk
Neomycin blood agar
Strict anaerobe
Clostridium difficile
Cytotoxicity test on human cell-line
ELISA to detect Clostridium perfringens exotoxin

Cycloserine cefoxitin fructose egg yolk agar (CCFA)
Vibrio Cholerae
Oxidase (+)
TSI (acidic slant acidic deep; no gas)
Agglutination of bacteria by polyvalent O1 antibody

TCBS (yellow colonies), Blood agar
Vibro parahemolyticus
Oxidase-positive
Grow in 10% NaCl

TCBS (green colonies)
Bacillus cereus
Lecithinase (+)
Motile (swarming on agar)
Catalase (+)
Indole (-)
Nitrate (+)
Urease (-)
Citrate (+)
MR (-)
VP(+)
TSI (Alkaline slant, acidic deep)
Oxidase (-)
Lactose fermenting; sucrose non-fermenting

Sheep’s blood Agar (beta hemolytic), large greenish colonies),
Mannitol (grey colonies)
Yersinia enterocolitica
Motile at 25 deg
MR (+)
VP (-)
Urease (-)
Indole (-)
Citrate (-)
Catalase (+)
TSI (acidic slant acidic deep; no gas)

MacConkey Agar (small lactose negative colonies)
EMB
Staphylococci aureus
Catalase (+)
Citrate (-)
Coagulase (+)
Indole (-)ME (+)
Oxidase (-)
Urease (-)
VP (-)
TSI (acidic slant acidic deep)

Sheep blood agar (beta hemolytic, yellow colonies)
Mannitol salt agar (yellow colonies)
7.5% NaCl media

Done by Kent and Ye Tun


CASE 3

E. coli Oxidase -
Catalase +
Indole +
Methyl red +
Voges-Prokauer -
Citrate Utilization -
Urease -
TSI; H2S production Acid slant/ acid butt & gas produced;
+
MacConkey agar Lactose fermenters; (red colonies)
Blood agar β-hemolytic
Klebsiella sp (Klebsiella pneumuniae, Klebsiella oxytoca) Oxidase -
Catalase +
Indole +
Methyl red -
Voges-Prokauer +
Citrate Utilization +
Urease -
TSI; H2S production Alkaline slant/ acid butt;
-
MacConkey agar Lactose fermenters; (pink colonies)
Blood agar Mucoid colonies formed
Enterobacter (Enterobacter aerogenes) Oxidase -
Catalase +
Indole -
Methyl red -
Voges-Prokauer +
Citrate Utilization +
Urease
TSI; H2S production
MacConkey agar
Blood agar
Serratia Oxidase -
Catalase +
Indole
Methyl red -
Voges-Prokauer +
Citrate Utilization +
Urease
TSI; H2S production
MacConkey agar
Blood agar
Proteus mirabilis Oxidase -
Catalase +
Indole +
Methyl red +
Voges-Prokauer -
Citrate Utilization
Urease +
TSI; H2S production Alkaline slant/ acid butt;
+
MacConkey agar Non-lactose fermenters; (white colonies)
Blood agar Swarming observed
Morganella morganii Oxidase -
Catalase +
Indole
Methyl red
Voges-Prokauer
Citrate Utilization
Urease +
TSI; H2S production
MacConkey agar Non-lactose fermenters; (white colonies)
Blood agar
Pseudomona aeruginosa Oxidase +
Catalase +
Indole -
Methyl red
Voges-Prokauer
Citrate Utilization +
Urease
Coagulase
TSI; H2S production
MacConkey agar Non-lactose fermenters; (white colonies)
Blood agar
Staphylococcus aureus
Catalase +
Methyl red
Voges-Prokauer
Citrate Utilization
Coagulase +
MacConkey agar -
Blood agar β-hemolytic
Staphylococcus saprophyticus
Catalase +
Methyl red
Voges-Prokauer
Citrate Utilization
Urease +
Coagulase -
MacConkey agar Non-lactose fermenters; (white colonies)
Blood agar -
Enterococcus sp
Catalase
Indole
Methyl red
Voges-Prokauer
Citrate Utilization
Urease
Coagulase
TSI; H2S production
MacConkey agar
Blood agar

Done by Joan


CASE 4

Possible Organisms Biochemical Tests & Results Culture agars
Streptococcus pneumoniae
Gram Staining +ve
Catalase -ve

2. 5% Sheep Blood agar with Optocin sensitivity Alpha-hemolysis with sensitivity to optocin
Klebsiella-Enterobacter-Serratia (Klebsiella sp.)
Gram Staining -ve
Oxidase -ve
Indole +ve/-ve
MR-VP +/- or -/+
Citrate +ve
H2S Production -ve
Lysine Decarbocylase +ve

Lactose Fermentation (MacConkey agar) +ve
Bordetella pertussis
Gram Staining -ve
Oxidase +ve
Urease -ve
Nitrase -ve
Citrate -ve

Borde+ - Gengou agar
BCYE agar

Done by Charmaine


CASE 5

Staphylococcus aureus
Gram Staining +
Catalase +
Coagulase +

Mannitol Salt Agar with 7-9% NaCl Mannitol fermentation
Sheep Blood Agar Hemolysis of blood agar
Streptococcus pyogenes
Gram Staining +
Catalase -
PYR (Pyrrolidonearylamidase) Test +

Sheep Blood Agar Beta Hemolysis
Enterococci
Gram Staining +
Bile- esculin test -

Sheep Blood Agar Gamma Hemolysis
6.5% NaCl Agar Can grow with 6.5% Nacl
Escherichia coli
Gram Staining -
Indole +
Methyl Red +
Voges Prokauer -
Citrate -
TSI Acid slant and butt with gas production but no H2S in TSI

MacConkey Lactose fermentative (deep red colony)
Eosin Methylene Blue( EMB) Green metallic sheen on EMB
Pseudomonas aeruginosa
Gram Staining -
Oxidase +
Catalase +
Indole +
Citrate +
Urease -
TSI -
Pyocyanin -
Fluroscein -

MAC Non- lactose fermentative (colourless colony on MAC)
Pseudo F agar PositiveFluorescein pigment in Pseudo F agar

Done by Ai Tee


CASE 6
Gardnerella vaginalis
Catalase -
Oxidase -
Vaginal pH test pH of vaginal fluid >5.0
Whiff test Release of bad-smelling odor when discharge is mixed with KOH
DNA Probe (Affirm™ VP III) High concentrations of G. vaginalis in VP III test

Chocolate agar Small, circular, convex, gray colonies
HBT agar
Colistin-oxolinic acid blood agar Beta-hemolysis
E. coli
Triple Sugar Iron (TSI) test TSI test show acid slant, acid butt with gas.
Indole test +
Methyl red test +
Voges-Proskauer (VP) test -
Citrate test -

EMB agar Black colonies with greenish-black metallic sheen on EMB agar
MacConkey agar Deep red colonies on MacConkey agar (lactose fermentor)
TSI agar
Pseudomonas aeruginosa
Oxidase test +
Catalase test +
Nitrase tese +
Lipase test +
TSI test Metallic sheen growth on the surface of TSI agar

EMB agar
Mac Conkey’s agar (sterile specimen)
Cetrimide agar (non-sterile specimen) Colonies appear flat, large, and oval. Secretes blue-green pigment (pyocyanin) on Cetrimide agar
TSI agar No colour change on TSI medium (K/K/g-/H2S- profile)
Neisseria gonorrhoeae
Oxidase test +
Direct immunofluorescence

Thayer-Martin chocolate agar Growth of spherical shape colonies on TM chocolate agar
Chlamydia Trachomatis
Direct immunofluorescence Presence of monoclonal antibodies (mAbs) to epitopes in the VS4 region of MOMP
ELISA Presence of Chlamydia antigens in ELISA test
PCR
DNA-based tests.

Blood agar
Mac Conkey’s agar
Trichomonas vaginalis
Vaginal pH test pH of vaginal fluid >4.5
PCR
Enzyme immunoassay Presence of T. vaginalis antigen in enzyme immunoassay
Direct Immunofluorescence.

Blood agar
Mac Conkey’s agar
Mobiluncus
Catalase test -
Indole test -
Oxidase test -

Blood agar Clear, colourless colonies, around 2mm.
Mac Conkey’s agar
Enterobacter-Klebsiella-Serrtia
-

Blood agar
Mac Conkey’s agar Deep red colonies on MacConkey agar (lactose fermentor)
Proteus-Providencia-Morganella
Urease test +
Indole test +

Blood agar Appear as swarming on blood agar.
Mac Conkey agar Colourless colonies on MacConkey agar (Non-lactose fermentor)
Enterococcus faecalis
Catalase test -

Blood agar Alpha, beta, or non-hemolytic on blood agar.
Mac Conkey agar
Candida albicans
-

Blood agar
Sabouraud’s agar Presence of white, butyrous colonies.
Staphylococcus saprophyticus
Catalase test +
Coagulase test -
Urease test +
Lipase test +

Blood agar
Mac Conkey’s agar Spherical, irregular grape-like cluster in culture.

Done by Adrian

Sunday, December 2, 2007

Group Post: MMIC dPBL 1

List of possible organisms for each case
Diagnosis Possible bacteria species Morphology & Microscopy
Case 1 Urinary Tract Infection (UTI)
Staphylococcus aureus

Staphylococcus saprophyticus

Enterococcus faecalis

Gram Positive, Spherical-shaped, immobile and form grape-like clusters

Gram Positive, Cocci-shaped occur singly and in pairs, short chains, and grape-like clusters

Gram Positive, Cocci-shaped in pairs and chains
Case 2 Enterocolitis
Campylobacter jejuni

Shigella species

Salmonella species

Escherichia coli (entero-pathogenic; EPEC)

Clostridium perfringens

Clostridium difficile

Vibrio Cholerae

Vibro parahemolyticus

Bacillus cereus

Yersinia enterocolitica

Staphylococci aureus

Gram-negative rods

Gram-negative rods

Gram-negative rods

Gram-negative rods

Gram-positive rods

Gram-positive rods

Gram-negative rods

Gram-negative rods

Gram-positive rods

Gram-negative rods

Gram-positive cocci
Case 3 Urinary Tract Infection (UTI)
E. coli

Klebsiella sp (Klebsiella pneumuniae, Klebsiella oxytoca)

Enterobacter (Enterobacter aerogenes)

Serratia

Proteus mirabilis

Morganella morganii

Pseudomona aeruginosa

Staphylococcus aureus

Staphylococcus saprophyticus

Enterococcus sp.

Gram negative bacilli

Gram negative bacilli

Gram negative bacilli

Gram negative bacilli

Gram negative bacilli

Gram negative bacilli

Gram negative bacilli

Gram positive cocci

Gram positive cocci

Gram positive cocci
Case 4 Bronchitis
Strep. Pneumoniae

Klebsiella-Enterobacter-Serratia (Klebsiella sp.)

Bordetella pertussis

Gram positive, diplococci, lancet-shaped

Gram negative rods

Gram negative, coccibacilli
Case 5 Wound Infection
Beta Haemolytic Streptococci (Streptococcus pyogenes)

Enterococci (Enterococcus faecalis)

Staphylococci (Staphylococcus aureus/MRSA)

Clostridium

Pseudomonas aeruginosa

Enterobacter species

Escherichia coli

Klebsiella species

Proteus species

Bacteroides

Gram-positive facultative anaerobes cocci

Gram-positive facultative anaerobes cocci

Gram-positive facultative anaerobes cocci

Gram-positive anaerobe rod

Gram-negative aerobic rods

Gram-negative facultative rods

Gram-negative facultative rods

Gram-negative facultative rods

Gram-negative facultative rods

Gram negative Anaerobes rods
Case 6 Urinary Tract Infection (UTI)
Gardnerella vaginalis

Chlamydia trachomatis

Neisseria gonorrhoeae

Ureaplasma urealyticum

Staphylococcus saprophyticus

Enterobacteriaceae (E. coli, serratia, klebsiella, enterobacter, citrobacter)

Enterococci

Gram-negative rod with a gram-positive cell wall

Gram-negative bacteria, coccoid or rod-shaped

Gram negative cocci

Gram-negative bacteria

Gram positive, globular shaped, colonies resemble grape-like clusters

Gram-negative, rod-shaped

Gram-positive cocci which often occur in pairs (diplococci)
Authors: Lizzie, Kent & Ye Tun, Joan, Charmiane, Ai Tee and Adrian (according to case no.)


Brief description of diagnosis:


1. Urinary Tract Infection (UTI)


A urinary tract infection (UTI) is a bacterial infection that affects any part of the urinary tract. The urinary tract is made up of the kidneys, ureters, bladder, and urethra, and each plays a role in removing liquid waste from the body. Although urine contains a variety of fluids, salts, and waste products, it usually does not have bacteria in it. When bacteria get into the bladder or kidney and multiply in the urine, they cause a UTI.

Cystitis: bladder infection.

Pyelonephritis: kidney infection is much more serious.

Urethritis: urethra infection.

Each type of UTI may result in more specific signs and symptoms, depending on which part of your urinary tract is infected. The symptoms shown in acute pylonephritis are closest to what is given in the case study, whereby it is an infection of your kidneys may occur after spreading from an infection in your bladder. Kidney infection can cause upper back and flank pain, high fever, shaking chills, and nausea or vomiting.

Pyelonephritis is an inflammation of the kidney and upper urinary tract that usually results from noncontagious bacterial infection of the bladder (cystitis). In acute pylonephritis, bacteria begin colonising the tubules and connective tissue of the kidney itself. Small abscesses and streaks of pus begin to appear in the renal cortex and medulla respectively. Pyelonephritis most often occurs as a result of urinary tract infection, particularly in the presence of occasional or persistent backflow of urine from the bladder into the ureters or kidney pelvis (vesicoureteric reflux).

Urinary catheterization is the insertion of a catheter into a patient's bladder through the urethra, to drain urine from the bladder into an attached bag or container. Indwelling catheters should be restricted to patients whose incontinence is caused by urinary tract obstruction that can not be treated, and for which alternative therapy is not feasible. However, the catheter may introduce bacteria into the urethra and bladder, resulting in urinary tract
infection. The risk for developing a UTI increases when long-term catheterization is required.

Bacteria causing UTI may be isolated from the vaginal discharge. UTI can also result from sexual intercourse where bacteria from the vaginal area is transferred to the urethra and bladder.

Authors: Lizzie, Joan and Adrian


2. Enterocolitis


Enterocollitis is the inflammation of the small intestine and colon. Disease-causing bacteria usually invade the small intestines and colon and cause inflammation (blood or pus in the stool, fever) and abdominal pain and diarrhea. Campylobacter jejuni is the most common bacterium that causes acute enterocolitis in the U.S. Other bacteria that cause enterocolitis include Shigella, Salmonella and EPEC (E. Coli Enteropathogenic). These bacteria usually are acquired by drinking contaminated water or eating contaminated foods such as vegetables, poultry, and dairy products.

Enterocolitis caused by the bacterium Clostridium difficile is unusual because it often is caused by antibiotic treatment. It is also the most common nosocomial infection (infection acquired while in the hospital) to cause diarrhea.

E. coli O157:H7 produces a toxin that causes hemorrhagic enterocolitis (enterocolitis with bleeding). There was a famous outbreak of hemorrhagic enterocolitis in the U. S. traced to contaminated ground beef in hamburgers (hence it is also called hamburger colitis).

Bacterial overgrowth of the small intestine.

Because of small intestinal problems, normal colonic bacteria may spread from the colon and into the small intestine. When they do, they are in a position to digest food that the small intestine has not had time to digest and absorb. The mechanism that leads to the development of diarrhea in bacterial overgrowth is not known.

Authors: Kent and Ye Tun

3. Bronchitis


Bronchitis is an inflammation of the bronchi of the lungs. There are two main types of bronchitis: acute and chronic bronchitis.

Acute bronchitis can be caused by viruses such as influenza A and B, parainfluenza virus, respiratory syncytical virus, coronavirus, adenovirus, and rhinovirus. Bacterias are also able to cause acute bronchitis. Air pollutants such as cigarette smoke, dusts, or fumes of chemicals are also able to cause acute bronchitis.

The symptoms of acute bronchitis include:

* Sore throat

* Chest congestion

* Sinus fullness

* Breathlessness

* Wheezing

* Slight fever and chills

* Overall malaise

* Cough

Chronic bronchitis is where by the signs and symptoms become prolonged, and is defined clinically as a
persistent cough that produces sputum (phlegm) and mucus, for at least three months in two consecutive years. Chronic bronchitis is part of chronic obstructive pulmonary disease (COPD).

Author: Charmaine

4. Surgical Site Infection (SSI)


There are different levels of SSI:

• Superficial incisional, affecting the skin and subcutaneous tissue.

• Deep incisional, affecting the facial and muscle layers.

• Organ or space infection affecting any part of the anatomy opened or manipulated during the operation.

Symptoms: Pain, fever, inflammation, swelling and pus formation



Causes of infection:

-Microbes contamination (most commonly by S. aureus, S. pyogenes or P. aeruginosa

-Migration of patients’ own bacterial normal flora

-The materials or equipment used in the operational procedures (for e.g.: poor surgical techniques)

Author: Ai Tee

Wednesday, November 7, 2007

19th Week of SIP - Medical Microbiology

Hi All!

I am sharing about a very scary virus infection. It causes hepatitis and has mortality rate of 30 percent. Quite scary huh!

We all heard about hepatitis B. But the motality rate of hepatitis B is not that high (0.5 - 1%). However this particular virus is somehow related to hepatitis B virus and it depends on the hepatitis B virus to infect the host.


Hepatitis D Virus

This interesting virus is the Hepatitis D Virus (HDV). HDV is a single-stranded RNA virus and it has HDAg as a enveloped protein (enclosing the RNA) and HBsAg (derived from hepatitis B virus) as its surface antigens. Without the HBsAg coating, HDV cannot infect, replicate or express on its own. The famility of HDV has not been identified.

Route of transmission and symptoms are similar to those of HBV. But it is more severe. It was reported that 70-80% of chronic HBV carriers with HDV superinfection develope chronic liver disease wuth cirrhosis.

As I mentioned HDV need HBV, thus the disease occur when HDV either superinfects the chronic HBV carriers or coinfect the person together with HBV. Symptoms are more severe in superinfection cases.

There is no vaccine for HDV and the best way to prevent it is to avoid risk behaviors. Immunization to HBV can avoid coinfection.

Okay...so much about the background and now I am sharing the laboratory diagnosis of HDV.

HDV is diagnosed serologically by detecting total antibodies to HDAg (delta antigen) by Competitive ELISA. The principle of the essay is as below. Enjoy....



Anti-HD present in the sample and labelled anti-HD antibodies compete for a fixed number of HDAg on the surface (sold-phase). The enzyme-labelled anti-HD gives colour after adding substrate, and the concentration of enzyme-labelled anti-HD can be worked out in relation to the O.D. reading. The amount of labelled anti-HD measured is inversly propotional to the concentration of anti-HD present in the samples.


OK. That's all for my posting. I hope you all enjoy it.
All the best for your SIP & MP yeah!
Cheers
~Ye Tun
TG01

Tuesday, November 6, 2007

19th week of SIP- Lab Techniques( Research)

hi, time passes and we will be back to skool soon...
tis time, I am going to discuss about
cell transfection and cell lysis.

Cell transfection:

It is the introduction of a foreign gene into mammalian cultured cells. My main purpose of the cell transfection is to obtain the maximum protein yield from the cells.

Applications of cell transfection: to make proteins for clinical or research applications
Changing the protein expression profile of a cell to assay for the effects of a gene to study for the cell physiology, endogeneous proteins, phenotypes or over expression of the proteins and addition of genetic markers to a cell line
The cell transfection is done by introducing a maximum amount of 3 ug of DNA but it also varies between cell lines. The cell line that I am using is the HeLa cell and they can become confluent between a day. Once they reach 20 – 30 millions cells, we must not allow them to grow anymore or they can mutate since the HeLa cells are immortal.

Cell transfection can be accomplished through several methods, including microinjection of foreign DNA into the cell or through a chemical or biological reagent. However, in my lab, we only introduce the DNA using the chemical reagent, lipofectamine and opti-mem.

Procedures:

Cell transfection can only take place in an Antibiotic- free media. A day before transfection, 1.2 millions cells are aliquoted into a 60 mm dish and resuspended in an antibiotic-free media. Transfection in a 100mm dish is time consuming and uses more reagents and DNA, thus only 60mm dish is used for transfection.

After 8 hrs from the time of transfection, the old antibiotic- free media is replaced with complete media.
After 24-48 hrs, the cells are lysed, harvested and quantitated with Bradford assay.

Things to note before and after cell transfection :
the cell density and its appearance: Cells must reach 70-90% confluency during transfection and during lysis. Anything that is not within the range will cause the cells to die due to toxicity. Besides, we need to monitor for cell numbers after 24-48 hrs from the time of transfection as there might be cell death and we must make sure that the cells that are still alive is enough for the next step- cell lysis. Cell death can occur often in cell transfection due to the stress they undergo. Cell abnormalities must also be observed.

Cell lysis

After 24-48 hrs of ill- treating the cells with the foreign DNA and reagents, the cell are ready to be lysed!!! Hahahaha… cell lysis is done with the lysis buffer with protease inhibitor (PI). There are different ways of cell lysis and will be mentioned later.

Procedures:

Method 1: cell scrapping

The old media is removed. The dishes will be rinsed with phosphate buffer saline (PBS). Lysis buffer is pipetted into the dishes and cells are scrapped with a cell scrapper. The contents left inside the dishes are transferred into a new tube and left to stand on ice for 10 mins.
NB: After harvesting the cells out from the dishes, all tubes must be kept chill at 4C.


After the 10 mins of incubation, the tubes are spun at 13000 rpm for 10 mins at 4C.
After spinning, the supernatant is pipetted out into a new tube and we can quantitate the protein concentration for our pull down assay and load for Western Blot analysis. Normally, lysate from cell scrapping may gives a false high protein estimation.

Method 2: passing thru the syringe

The old media is removed. The dishes are trypsinized twice with 1 ml of trypsin and harvested with PBS and the PBS- resuspended cells are transferred into new tube. The tubes are spun for 5 mins at 6000rpm and the supernatant is removed. The remaining pellet is dipped into liquid nitrogen and then, thawed in ice. After which, the pellet is resuspended with lysis buffer. An insulin syringe will be used to puncture the cells through the syringe to create pressure for lysing the cells. The tubes are allowed to stand in ice for 10 mins and centrifuged for another 10 mins. Supernatant is removed and transferred into new tube and ready for quantitation.

Method 3: sonication

Instead of using an insulin syringe to lyse the cells, sonication can also be used to lyse the cells. the tubes are allowed to stand in ice for 10 mins and sonicated. after which, the tubes will be spun for 10 mins to harvest the cells. However, the number of pulses and how long it takes for each pulse is an important factor affecting on protein yield.

Thats all for my last blog for SIP!! take care..n enjoy last few days of work!!

Ai Tee
TG 01
0503160D

Wednesday, October 31, 2007

18th week of SIP - Lab Techniques (Research)

Hey! Last 2nd week of SIP now aye? Soon, it'll be over! =D

Anyway, I'll be touching on this assay called Rat/Mouse Insulin ELISA Assay

Rat/Mouse Insulin ELISA Assay

Insulin is a hormone made by the beta cells in the islets of Langerhans that regulates the level of glucose in the blood.

When blood glucose level increases, insulin will be produced and released into the blood circulation.

This picture gives an overview on how insulin regulates blood glucose level.

Taken from: here.

This assay is performed in vitro.

Principle:
The basis of this assay is through sandwich ELISA -

1. Binding of insulin molecules to the wells of a multi-well plate by a pre-titered amount of monoclonal mouse anti-rat antibodies, and bindin of biotinylated polyclonal antibodies to the captured insulin molecules.

2. Unbound materials are washed away.

3. Binding of horseradish peroxidase to the bound biotinylated polyclonal antibodies.

4. Unbound materials are washed away.

5. The antibody-enzyme conjugates are quantified by monitoring the activities of horseradish peroxidase in the presence of the substrate, 3,3',5,5'-tetramethylbenzidine.

6. The enzyme activity will be measured spectrophotometrically at 450nm. An increase concentration of insulin will lead to an increase formation of the blue color.



The more insulin molecules captured by the antibodies, the higher the absorbance.
Thus an increase in absorbance is directly proportional to the amount of captured insulin.

-------------------------------------------

Message from Mr. Poh:
Poster format -
1. Go to MS Powerpoint
2. Under "Page Setup", key in the height and width of A1 size
Height: 33.07"
Width: 23.38"
3. Type everything that is to be in the poster on just this ONE slide.

Price for printing A1 size poster in the school : $18. *faints*

That's all!
Charmaine Tan
TG01

Saturday, October 27, 2007

17th week of SIP - Laboratory Management

(This entry will get drier as it goes.....)


Since the school semester ended, our job as a TSO revolves around laboratory management and cleanliness! It's the best time to carry out 'spring cleaning" and organising paperwork! And it's maintenance of both MCT and MBIO lab! There're 2 more lab in MCT lab for those who took MCT before should know.. '4' labs down to me, charmaine and my poor supervisor to clean up.. (T_T) Chemicals such as media prepared by students were discarded. Contaminated media were autoclaved befor disposal. Equipment were cleaned with 70% alcohol or detergent. We had cleaned the incubators, water baths, 4 degrees fridge which we kept our DMEM for MCT practical (due to its size, we took one whole day to clean it and it looks exactly like a brand new fridge!) And the most shocking thing about the fridge was that the exhaust fan cover was transparent in colour! the amount of dust accumulated deserved 10 times "oh my god"! Latest news - all the labs had undergone big renovation! the lecturer's bench disappeared.. It's a pity we're not using MCT or MBIO lab anymore.. We know all the passwords and keys to everything! haha..

Other than spring cleaning, we had done tonnes of admin work! I guess we spent at least a month on the paperwork! Each lab have to maintain 4 types of documents. For File#1, it consists of general records like list of chemical, accessories and equipment (abbreviated LOC, LOA, LOE).. For the LOC we had to check if all the chemicals in the list are atill available.. For your information, there are 2 safety cabinet, 1 flammable cabinet, 4 4degree fridge, 4 -20degree fridge and 1 -80degree fridge! after checking every chemicals, our hands will be so freezing numb! And the checking was not only done once.. I suppose we can memorise where all the chemicals are... become walking chemical inventory list! Equipment were checked against the LOE.. As the LOA was updated quite recently, therefore we can leave it aside.. *phew*

For File#2, it contains all the MSDS! For this particular file, we spent almost 7 days on sorting the MSDS for two labs! Imagine 7 days 9-6 only MSDS! MSDS-phobia... As our supervisor just took over these 2 labs therefore the workload is heavier.. Lab file #3 basically contains all the maintenance records and service contracts for equipment. Lab file #4 is on the laboratory equipment SOPs and the inventory list. During LMQA, Ms Chew said before that our school has spent much money and alot of paperwork to get ISO 9001 & ISO 14001 acreditation.. Now I really know how much paperwork was required! It's an eye opener! So next time, we see any TSOs.. we must really appreciate them! on top of preparing reagents for us, they still have to do all the paperwork and maintaining a clean and organised lab!

Joan
TG01

Sunday, October 21, 2007

16 week of SIP- Urinary Stone Analysis


I.
Introduction













If you've ever passed a kidney stone, you're not likely to forget the experience — it can be excruciatingly painful. Kidney stones (renal lithiasis) are an ancient affliction dating back to the age of the Egyptian pyramids, yet they are still a common disorder today. The incidence of kidney stones has been increasing in recent decades. Although the reasons for this are still unclear, many experts believe that diet choices and lack of fluids are important factors that have contributed to this increase.

Your kidneys are two bean-shaped organs, each about the size of your fist. They're located in back of your abdomen on each side of your spine, and their main function is to remove excess fluid, unneeded electrolytes and waste from your blood in the form of urine. The ureters carry urine from your kidneys to your bladder, where it's stored until you eliminate it from your body.

Kidney stones usually form when your urine becomes too concentrated. This causes minerals and other substances in urine to form crystals on the inner surfaces of your kidneys. Over time, these crystals may combine to form a small, hard mass, or stone.



What a rock! Yup, its a kidney stone.







II. Signs and symptoms

A patient is not likely to have signs and symptoms unless a kidney stone is large, causes a blockage, is associated with an infection or is being passed. Then the most common symptom is an intense, colicky pain that may fluctuate in intensity over periods of five to 15 minutes. The pain usually starts in the patient’s back or side just under or below the edge of his ribs. As the stone moves down the ureter toward the patient’s bladder, the pain may radiate to his lower abdomen, groin and genital structures on that side. If the stone stops moving, the pain may stop too. Other signs and symptoms may include:

  • Bloody, cloudy or foul-smelling urine
  • Nausea and vomiting
  • Persistent urge to urinate
  • Fever and chills if an infection is present



The patient must try to catch the stone in a strainer during urination.













III. Causes

The crystals that lead to kidney stones are likely to form when the patient’s urine contains a high concentration of certain substances — especially calcium, oxalate, uric acid and rarely, cystine — or low levels of substances that help prevent crystal formation, such as citrate and magnesium. Crystals also may form if the patient’s urine becomes too concentrated or is too acidic or too alkaline.

A number of factors can cause changes in the patient’s urine, including the effects of heredity, diet, drugs, climate, lifestyle factors and certain medical conditions. Each of the four main types of kidney stones has a different cause:

  • Calcium stones

Roughly four out of five kidney stones are calcium stones. These stones are usually a combination of calcium and oxalate. Oxalate is a compound that occurs naturally in some fruits and vegetables. A number of factors can cause high concentrations of these substances in urine. Excess calcium, for instance, may result from ingesting large amounts of vitamin D, from treatment with thyroid hormones or certain diuretics, and from some cancers and kidney conditions. You may also have high levels of calcium if your parathyroid glands, which regulate calcium metabolism, are overactive (hyperparathyroidism). On the other hand, certain genetic factors, intestinal bypass surgery and a diet high in oxalic acid may cause excess amounts of oxalate in your body.

  • Struvite stones

Found more often in women than in men, struvite stones are almost always the result of chronic urinary tract infections caused by bacteria that produce specific enzymes. These enzymes increase the amount of ammonia in the urine, which is incorporated in the crystals of struvite stones. These stones are often large, may have a characteristic stag's-horn shape and can seriously damage the patient’s kidneys.

  • Uric acid stones

These stones are formed of uric acid, a byproduct of protein metabolism. A patient is more likely to develop uric acid stones if he has undergone chemotherapy, consumes a high-protein diet or has certain genetic factors that predispose him to the condition.

  • Cystine stones

These stones represent only a small percentage of kidney stones. They form in people with a hereditary disorder that causes the kidneys to excrete excessive amounts of certain amino acids (cystinuria).


Kidney stones vary in size and shape.


Golf-ball sized and round




Small and smooth








Jagged and yellow






IV. Material & Methods

Sample Preparation

Dissolve a sample as homogenously as possible of the urinary calculus to be analysed. From this solution the various components of the calculus are determined semi-quantitatively, the titrimetric method being used for calcium and a colorimetric method (ie. visual colour comparison) being used for oxalate, phosphate, magnesium, ammonium, uric acid and cysteine. The composition of the urinary calculus is obtained form the results of these determinations with the help of the test kit’s calculation aid.

1. 1. Finely triturate the calculus to be analysed in a motar.

2. 2. Mix the resultant powder thoroughly and using a spatula, transfer a tipful to a plastic boat.

3. 3. Add 5 drops of Sulfuric acid. Stir with spatula to ensure complete dissociation.

4. (Evolution of gas during dissolution indicates carbonate.)

5. 4. Transfer the solution into a 100ml graduate filed to one-third with distilled water.

6. 5. Make up to the 50ml mark with distilled water and mix well with the plastic boat.

7. 6. Transfer 5ml of the sample solution into each of several test tubes for other calculi composition testing, with the exception of Magnesium.

8. 7. For Magnesium testing, transfer 1 ml of the sample solution into another test tube with 4 ml DI water.

Individual calculi composition analysis

  1. Calcium

Reagents

Reagent 2: Sodium hydroxide solution 27%

Reagent 3: Calconcarboxylic acid tituration

Reagent 4: Titriplex III solution

Procedure

To the sample solution, add 2 drops of reagent 2 and one spatulaful of Reagent 3 and shake.

Continue shaking, and while doing so add reagent 4 drop by drop until the colour of the solution changes from red to blue. Count the drops required for the colour change to occur.

The number of drops required multiplied by 5 gives the percentage calcium content of the calculus.


  1. Oxalate

Reagents

Reagent 5: Borate buffer solution

Reagent 6: Iron (III) chloride solution

Reagent 7: Sulfosalicylic acid solution

Procedure

To the sample solution, add subsequently while shaking

2 drops of Reagent 5,

2 drops of Reagent 6,

3 drops of Reagent 7; allow to stand for 2 minutes.

Compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage oxalate content of the calculus.






  1. Ammonium

Reagents

Reagent 8: Potassium tetraiodomercurate (II)

Reagent 2: Sodium hydroxide solution 27%

Procedure

Add subsequently to the sample solution, while shaking,

3 drops of Reagent 8 and

3 drops of Reagent 2.

Compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage ammonium content of the calculus.









  1. Phosphate

Reagents

Reagent 9: Ammonium molybdate solution

Reagent 10: Reducing solution 4: methylaminophenol sulfayte soldium disulfite)

Procedure

Add subsequently to the sample solution, while shaking

5 drops of Reagent 9 and

5 drops of Reagent 10; Allow to stand for 5 minutes

Compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage phosphate content of the calculus.






  1. Magnesium

Reagents

Reagent 11: Buffer solution (borate buffer)

Reagent 12: Colour reagent (1-azo-2-hydroxy-3-(2, 4-dimethyl-carboxanilido)-napthalene-1’-2-hydroxylbenzene-5-sodium sulfonate) solution

Procedure

Pipette 1 ml of sample solution into a reaction vessel and make up to the calibration mark with distilled water. Add 10 drops of Reagent 11 and 10 drops of Reagent 12 while shaking.

After 1 minute, compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process. Read off the percentage magnesium content of the calculus.








  1. Uric Acid

Reagents

Reagent 13: Molybdatophosphoric acid solution

Reagent 5 Borate buffer solution

Procedure

Add 3 drops of Reagent 13 to the sample solution, shake, and allow to stand for 2 minutes. Then add 2 drops of Reagent 5 and shake. Immediately compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process. The colour comparison should be performed within 10 seconds after the addition of Reagent 5 to the sample solution because the colour is not stable and liable to change to blue.

Read off the percentage uric acid content of the calculus.








  1. Cystine

Reagents

Reagent 14: Ammonia solution

Reagent 15: Reducing agent (sodium sulphite)

Reagent 16: Sodium nitroprusside titruation

Procedure

To the sample solution, add 10 drops of Reagent 14 and a red dosing spoonful of Reagent 15, and swirl until dissolution is obtained. 1 minute after the addition of Reagent 15, add a black dosing-spoonful of Reagent 16, and again shake until dissolution is obtained. Compare the colour of the solution in the reaction vessel with the appropriate colour scale 30 seconds after the addition of reagent 16, and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage cystine content of the calculus.









Result Reporting

Record down the percentage values for each of the calculi components on a result slip.


V. Conclusion


Urinary calculi analysis may sound tedious, but its actually very fun to perform....if there isn't much samples. Its like O'Level Chemistry practical..Qualitative Analysis. Hope it rings a bell.

I'm sure u got lotsa questions to ask. Just show some mercy ok? This topic is really vast. This message goes out especially to those from my workplace. . =P

Will upload more exciting photos soon. Stay tuned!


Kent Lieow
TG 01
0503261J


Sunday, October 14, 2007

15th Week of SIP - Medical Microbiology

Hi everyone,

Sorry for the late posting, it's me again! I am currently attached to the microbiology department! For the last week in the Department, my section head give me a mini test. Sound scary but fun...

Identification of Bacteria Species in Urine Culture

A urine culture may be ordered when symptoms indicate the possibility of a urinary tract infection, such as pain and burning when urinating and frequent urge to urinate. Antibiotic therapy may be prescribed without requiring a urine culture for symptomatic young women, who have an uncomplicated lower urinary tract infection. If there is suspicion of a complicated infection, or symptoms do not respond to initial therapy, then a culture of the urine is recommended.

Step 1: Gram Stain

Principle: A method of differentiating Gram Positive and Gram Negative bacteria based on the properties of the cell wall. Gram Positive bacteria's cell wall has a stronger attraction for crystal violet. Gram iodine is known as a MORDANT which will form a complex with crystal violet and will retain the crystal violet stain after washing with alcohol. Gram negative bacteria will become colorless after washing with alcohol, counterstaining with safranin will make them pink.

Procedure:
  1. Emulsify a colony of the bacteria (from the culture plate) into a drop of saline on a clean microscope slide.
  2. Heat fix the slide.
  3. Flood the slide with crystal violet for 1 min. Wash with running tap water.
  4. Flood with Gram's iodine for 1 min. Wash with running tap water.
  5. Carefully decolourize with 95% ethanol.
  6. Flood with safranin 1 min. Wash with running tap water.
  7. Look at the slide under microscope.
Result: Gram Negative - Pink, Gram Positive – Purple

For Gram Positive bacteria,

Step 2: Catalase Test

Principle: To identify which organism produces catalase enzyme that convert hydrogen peroxide to water and oxygen gas. Catalase enzyme help to protect bacterial cell wall against hydrogen peroxide which is a highly reactive compound. It is useful in differentiating Staphylococci and Streptococci. Catalase positive will produce bubble which is an indication of presence of Staphylococci.

Procedure: Simply place a loopful of cells from isolated colonies into a drop of 3% hydrogen peroxide on a clean microscope.

Result: Immediate generation of bubbles - Positive (Staphylococci), no bubbles - Negative (Streptococci).

Step 3: Coagulase test

Principle: This test is to identify the presence of bound coagulase or clumping factor which will attach to the cell wall of the bacteria. Bound coagulase reacts with fibrinogen in plasma causing the fibrinogen to agglutinate which will give a positive result. This test is useful in differentiating S. aureus from Catalase positive Staphylococci.

Procedure: Simply add a colony into a drop of thawed rabbit plasma and mix them thoroughly.

Result: Agglutination – Positive (S. aureus), No agglutination – Negative (Staphylococci)

Step 4: Sensitivity Plate and Purity Plate

Principle: Sensitivity Plate is to determine the effectiveness of antibiotic against micro-organism that has been isolate from the culture. Purity Plate is to check for the morphology of the bacteria growth on the plate to tally with the result found.

Procedure:
Sensitivity Plate:
  1. Emulsify half a colony from the culture plate into the saline till 0.5 McFarland turbidity.
  2. Lawn the whole MH agar plate and ¼ of 5% NaCl agar plate with the micro-organism.
  3. Place the antibiotic disks: VA, E, SXT, TE, FT, P, AM, CIP, AMC, FOS, FT on MH agar plate and OX on 5% NaCl agar plate.
Purity Plate:Streak the plate.

Result: For Sensitivity Plate, sensitive – antibiotic is effective, micro-organism grow far away from it, intermediate – antibiotic may or may not be effective and resistant – the antibiotic is ineffective, micro-organism grow around it.

For Gram Negative Bacteria

Step 2: Oxidase test

Principle: To identify organism that produce enzyme cytochrome oxidase. Cytochrome oxidase participates in the electron transport chain by transferring electron from donor molecule to oxygen. The oxidase test reagent contain reducing agent which is a compound that change colour when become oxidized.

Procedure: Simple smear an isolated colony onto the oxidase card

Result: Change of colour – positive, no change of colour - negative

Step 3: Microgen GN-ID

Principle: To identify the complete range of Enterobacteriaceae and other non-fastidious, oxidase positive and negative, gram negative bacilli. Microgen GN-ID comprises of 2 separate, 12 substrates, microwell strips, GN-A and GN-B. GN-A can be used alone and identifies oxidase negative, non-fastidious gram negative bacilli while GN-B needs to be used with GN-A, identifies the complete set of Enterobacteriaceae and other non-fastidious, oxidase positive and negative, gram negative bacilli. The wells contain dehydrated biochemical substrates that are reconstitute with a saline suspension of the organism to be identified. If the substrate is metabolized by the organism, colour change will occur during incubation or after adding addition of reagents.

Procedure:
  1. Perform oxidase test on the isolate prior to strip inoculation.
  2. Emulsify a single colony from the culture in sterile saline.
  3. Mix thoroughly.
  4. Add 3-4 drops of suspension to each well of the strip.
  5. Overlay specific will with 1 drop of mineral oil on the black circles.
  6. Seal the top of the microwell strip with adhesive strip and incubate at 37oC for 18-24 hours.
  7. Add a drop Indole Kovas, VP I and II and TDA to the respective well.
Result: Compare the colour shown on the microwell with the colour chart provides by the supplier and type in the result into the software provided by the supplier to identify the organism.

Step 4: Sensitivity Plate and Purity Plate

Principle: Sensitivity Plate is to determine the effectiveness of antibiotic against micro-organism that has been isolate from the culture. Purity Plate is to check for the morphology of the bacteria growth on the plate to tally with the result found.
Procedure:
Sensitivity Plate:
  1. Emulsify half a colony from the culture plate into the saline till 0.5 McFarland turbidity.
  2. Lawn the whole MH agar plate and ¼ of 5% NaCl agar plate with the micro-organism.
  3. Place the antibiotic disks: CRO, CXM, AM, SAM, GM, CIP, FT, SXT, FOS, AMC on blood agar plate.
Purity Plate: Streak the plate.

Result: For Sensitivity Plate, sensitive – antibiotic is effective, micro-organism grow far away from it, intermediate – antibiotic may or may not be effective and resistant – the antibiotic is ineffective, micro-organism grow around it.

Clinical Significance:

It is used to diagnose Urinary Tract Infection (UTI). It is done to determine the type of bacteria in the urine and the appropriate antibiotic for treatment. A UTI is an infection of one or more components of the urinary tract. UTI occurs at any age but women are much more likely than men to have them.

Hope that you all have learnt something after reading my blog. Feel free to ask questions.

Lizzie Chew (0503194C) TG01

Saturday, September 29, 2007

14th Week of SIP - Microbiology/Immunology

Hi all!

This is time for my sharing again. As I am currently attached to viral serology lab, I am learning different serological tests such as various enzyme immunoassays (EIA), hemeagglutination-inhibition tests (HI) and other specialized tests like immunoblots.


::Dengue In Focus

Since there were quit a number of dengue cases recently in Singapore, I think it would be useful to share how dengue is diagnosed.

Dengue virus can be isolated in the mosquito cell culture (C636 cell-lines) and specific dengue serotype can be identified using immunofluorescence method. Due to its specificity dengue isolation is the gold standard for dengue diagnosis. However viral isolation takes about 2 weeks.

Due to varies in its severity and undifferentiated symptoms, it is very important to diagnose dengue as early as possible and give appropriate treatments.

Therefore serodiagnosis of dengue which can detect dengue infection rapidly becomes the most common method. In this blog, I will be sharing about Dengue IgM Capture Enzyme-linked Immunosorbent Assay (ELISA).


::Dengue IgM ELISA


Principle


This particular dengue IgM kit uses Capture Enzyme-linked Immunosorbent method and specific dengue IgM is captured between anti-human IgM and commercial dengue antigen. The picture below clearly explains:


If O.D. reading is more than 1, it is considered as dengue IgM positive. Positive and negative control and calibrator added in each run for quality control. The assay is run 100% manually and takes about 4 hours.

Clinical Significance

Presence of dengue IgM shows that the patient is suffering from acute dengue infection as IgM are produced during the acute infection.


This test however cannot tell if the dengue infection is primary or secondary. Primary means this is the first time the patient is experiencing dengue infection. Secondary infection means patient had dengue before, but this time he/she is infected by dengue virus of different serotype from the first one.


Hemeagglutination-inhibition test is the only test which can determine the dengue infection is primary or secondary, acute, recent or recovering.

There are also commercially availiable rapid kits which uses various principles such as immunoblot, immunochromatographic, etc.

Well, I think this would be enough for this posting. Feel free to ask me if you have anything to clarify or wanna know more about dengue. All are welcome!

Thank you for reading. All the best for your SIP...6 weeks more yeah!

Cheers

Ye Tun
~TG01

Friday, September 21, 2007

13th week of SIP- Lab Techniques( Research)

Hi all, i think i miss out on my turn to blog one of those weeks.. so its ME again..
This time I m going to share with you a new assay I learnt: Co- Immunoprecipitation assay or in short, pull- down assay.

This assay works likes a protein- antibody reaction and with this assay, we can deduce whether are 2 proteins interacting with one another based on the Western Blot (WB) analysis which will be done after this.
For e.g: I wan to study if analog A binds to protein Z. normally, we will tag the analog A and protein Z with something so that we can detect them. Protein Z is tagged with the FLAG tag while HA tag for the TRX- analog and TRX proteins.
The analog A is attached to this protein scaffold (thioredoxine or TRX) to stabilize the structure.

I will have 3 reaction samples:
1. (-) Ctrl: Vector + Protein Z
2. (-) Ctrl: TRX + Protein Z
3. TRX- analog A + Protein Z
All this three DNA samples will be transfected into HeLa cells and then, we will lyse the Hela Cells to get the proteins translated.

After we lyse the cells and the cell Lysate are ready, we will save an aliquot mixed with protein dye without reducing agent for WB. The remaining will be topped up to 600- 1000ul with the lysis buffer.

Next, we will utilize beads. There are 2 types of beads: Protein G and A. however, I m using only the protein G. the protein G is attached to a solid matrix like agarose or sepharose and can bind to Ab. the beads will help to pulldown ab- protein complexes and when eluted, the complexes will detach from the beads and ready to load for WB analysis. (NB: Beads are hard to handle!!! So small and tiny and must be very careful not to suck them out in each step!)
Procedures:

Firstly, the beads will be pre-washed .
The lysate are then added to our pre-washed beads, and rotated at 4 °C for 1 hr. This step is called pre- clearing. We want to clear away those proteins in the lysate that will have an affinity to bind to the beads which can give non- specific bands under WB.
After that, the beads are spun down and the supernatant are transferred to another newly washed beads coupled with the Ab and rotated overnight at 4°C.
The next day, the beads are washed 5 times with wash buffer and eluted with 1 X TBS and protein dye. Proteins complexes that are bound together to the beads will therefore detach from the beads and can be loaded for WB analysis.

This assay often gives messy WB results. If we use a rabbit Ab for pulldown, we must use the mouse Ab for WB primary Ab and vice versa. The rabbit Ab will always give more background signal from WB than the mouse. Normally, I will also load the Lysate besides my pulldown in my WB. This allows me to compare and also troubleshoot whether my proteins are expressed in the Lysate or not. If my proteins expression is not detected in my lysate but I have a signal in my pulldown, it means that there might be something wrong in the experiment that can react to give a signal even my proteins are not around.
Any interaction can be seen based on the WB results and checked accordingly with the control to confirm that the experiment is working well and not due to any errors.
The success of pull down and protein expression will also depend upon the cell density for transfection and amount of DNA introduced into the cells.
So that’s all for the beads session. Feel free to ask me anything.

Hope you all are enjoying yourselves!!!

Ai Tee
TG 01
0503160D

Sunday, September 16, 2007

12th Week of SIP- Microbiology

Hey all!

It’s my turn to blog again. This time, I will post on something I have learned in the Microbiology lab. It is the testing and detection of Neisseria Gonorrhoeae.


What is Neisseria Gonorrhoeae?

Neisseria gonorrhoeae
(also known as Gonococci) is a species of Gram-negative bacteria responsible for the sexually transmitted disease Gonorrhea.


How is it detected/tested?

  • To test for Neisseria gonorrhoeae, an endocervical swab is taken from the patient, then plated.
  • Neisseria species is isolated from the primary culture by plating on a TM plate (selective media).
  • Perform a gram-stain. Look for gram negative diplococcus (Neisseria gonorrhoeae) under the microscope.
  • To further test for the presence of Neisseria gonorrhoeae, Bacticard Neisseria commercial test kit is used.


Principle of Bacticard Neisseria:

  • It is used for the identification of pathogenic Neisseria species
  • Kit utilizes 4 chromogenic substrates impregnated on individual test circles to detect preformed enzymes associated with pathogenic Neisseria.
  • The enzymes b-galactosidase and butyrate esterase hydrolyze the substrates 5-bromo-4-chloro-3-indolyl-b-D-galactoside and 5-bromo-4-chloro-3-indolyl butyrate respectively to form a blue-green complex in the presence of oxygen.
  • Gamma-glutamyl aminopeptidase hydrolyzes gamma-glutanyl-b-naphthylamide and prolylaminopeptidase hydrolyzes L-proline-b-naphthylamide and reacts with the Bacticard Neisseria Colour Developer to form a red colour.


Procedure for using Bacticard Neisseria:

  • Rehydrate each test circle with 1 drop of Bacticard Neisseria Rehydrating Fluid.
  • Smear several colonies of the organism to be tested across each test circle.
  • Incubate at room temperature for 2 mins.
  • Observe IB test circle for a blue-green colour, indicating a positive test. If positive, proceed to identification. If negative, continue incubation for additional 13 mins.
  • After incubation, observe BGAL test circle for a blue-green colour indicating a positive test. If positive, proceed to identification. If negative, proceed by adding 1 drop of Colour Developer to the GLUT and PRO test circles.
  • Observe for the development of a pink to red colour at 30 secs. Proceed to identification.


Interpretation of results:

    • IB test
      • Positive - Colour change to blue-green after 2 mins.
      • Negative - No colour change.
    • BGAL test:
      • Positive - Colour change to blue-green within 15 mins.
      • Negative - No colour change.
    • GLUT and PRO test:
      • Positive - Colour change to pink or red within 30 secs after addition of Colour Developer.
      • Negative - No colour change.
    • Neisseria gonorrhoea should show negative for IB, BGAL and GLUT tests, but positive for PRO test.

That’s all folks! Continue to enjoy your SIP while it last.

Out.


Adrian Tan
TG01
0503205G