77th XiaoBianTai's Medical Street

MMic dPBL-2: Problem Statement

2008-01-22T23:59:00.000+08:00

Problem Statement

There are outbreaks of viral, fungal and protozoa diseases among platoons of army soldiers in Indonesia. Soldiers reported sick after 2 weeks of jungle warfare training. It is of concern to the ministry that there are also sporadic reports of avian flu in the nearby villages. In view of these outbreaks, you have been tasked to conduct a pre-mission briefing with blogs and poster to educate future batches of soldiers.

Your group needs to keep a blog containing information about a variety of pathogens that cause problems during jungle training so that the soldiers are made aware of the dangers and could take the necessary precautions.

MMic dPBL-2: Possible Viral Infections

2008-01-22T16:22:00.000+08:00

References:

1. DPDx. (2008). Balantidiasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Dengue Fever > Dengue Fever

2. DPDx. (2008). Isospora belli. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Japanese Encephalitis> Japanese Encephalitis

3. DPDx. (2008). Cryptosporidium. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Tick-borne Encephalitis > Tick-borne Encephalitis

4. DPDx. (2008). Trichinellosis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hepatitis> Hepatitis

5. DPDx. (2008). Ascariasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Rabies > Rabies

6. DPDx. (2008). Ascariasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm > Search: Avian Flu> Avian Flu

7. DPDx. (2008). Ascariasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm > Search: Chikungunya > Chikungunya

Done by: Lizzie, Kent, Gail

MMic dPBL-2: Possible Fungal Infections

2008-01-22T16:15:00.000+08:00

(Molds)

(Yeasts)

Subcutaneous Mycoses

Systemic Mycoses

References:

Brooks GF, Carroll KC, Butel JS & Morse SA. 2007. Jawetz, Melnick & Adelberg’s Medical Microbiology, 24th Edition. The McGraw-Hill Companies, Inc.: pages 626-630, 646-648, 650-654.

Gillespie S & Bamford K. 2000. Medical Microbiology and Infection at a Glance. Blackwell Science: pages 70 – 73.

Levinson, W. (2004). Medical Microbiology & Immunology: Examination & Board Review, Eight Edition. The McGraw-Hill Companies, Inc.: pages 331-337.

Done by: Joan Lim & Ye Tun

MMic dPBL-2: Possible Protozoa Infections

2008-01-22T16:00:00.000+08:00

References:

1. DPDx. (2008). Balantidiasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Balantidiasis > Balantidiasis

2. DPDx. (2008). Isospora belli. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Isospora belli > Isosporiasis

3. DPDx. (2008). Cryptosporidium. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Cryptosporidium > Cryptosporidiosis

4. DPDx. (2008). Trichinellosis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Trichinellosis > Trichinellosis

5. DPDx. (2008). Ascariasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Ascariasis > Ascariasis

6. DPDx. (2008). Hookworms. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hookworms > Hookworms

7. DPDx. (2008). Trichuriasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hookworms > Trichuriasis

8. DPDx. (2008). Filariasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hookworms > Filariasis

9. DPDx. (2008). Hymenolepiasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hookworms > Hymenolepiasis

10. DPDx. (2008). Schistosomiasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hookworms > Schistosomiasis

11. DPDx. (2008). Opisthorchiasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hookworms > Opisthorchiasis

12. DPDx. (2008). Paragonimiasis. Retrieved January 21, 2008, from http://www.dpd.cdc.gov/dpdx/Default.htm >Search: Hookworms > Paragonimiasis

13. GSBS UTMB. (2007). Cestodes. Retrieved January 22, 2008 from http://gsbs.utmb.edu/microbook/ > Table of Contents > Cestodes

14. GSBS UTMB. (2007). Trematodes. Retrieved January 22, 2008 from http://gsbs.utmb.edu/microbook/ > Table of Contents > Trematodes

15. UALBANY School of Public Health. (2008). Helminths (Worms). Retrieved January 22, 2008, from http://www.albany.edu/sph/coned/ > Search > Helminths

Done by: Adrian Tan & Charmaine Tan

Group Post: MMIC dPBL-1

2007-12-09T01:52:00.000+08:00

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

Group Post: MMIC dPBL 1

2007-12-02T14:10:00.000+08:00

**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

19th Week of SIP - Medical Microbiology

2007-11-07T23:06:00.000+08:00

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

19th week of SIP- Lab Techniques( Research)

2007-11-06T21:48:00.000+08:00

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

18th week of SIP - Lab Techniques (Research)

2007-10-31T09:31:00.000+08:00

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

17th week of SIP - Laboratory Management

2007-10-27T20:52:00.000+08:00

(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

16 week of SIP- Urinary Stone Analysis

2007-10-21T22:23:00.001+08:00

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

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.

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.

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.

Read off the percentage ammonium content of the calculus.

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

Read off the percentage phosphate content of the calculus.

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.

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.

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

15th Week of SIP - Medical Microbiology

2007-10-14T23:27:00.000+08:00

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:

Emulsify a colony of the bacteria (from the culture plate) into a drop of saline on a clean microscope slide.
Heat fix the slide.
Flood the slide with crystal violet for 1 min. Wash with running tap water.
Flood with Gram's iodine for 1 min. Wash with running tap water.
Carefully decolourize with 95% ethanol.
Flood with safranin 1 min. Wash with running tap water.
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:

Emulsify half a colony from the culture plate into the saline till 0.5 McFarland turbidity.
Lawn the whole MH agar plate and ¼ of 5% NaCl agar plate with the micro-organism.
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:

Perform oxidase test on the isolate prior to strip inoculation.
Emulsify a single colony from the culture in sterile saline.
Mix thoroughly.
Add 3-4 drops of suspension to each well of the strip.
Overlay specific will with 1 drop of mineral oil on the black circles.
Seal the top of the microwell strip with adhesive strip and incubate at 37oC for 18-24 hours.
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:

Emulsify half a colony from the culture plate into the saline till 0.5 McFarland turbidity.
Lawn the whole MH agar plate and ¼ of 5% NaCl agar plate with the micro-organism.
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

14th Week of SIP - Microbiology/Immunology

2007-09-29T19:54:00.000+08:00

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

13th week of SIP- Lab Techniques( Research)

2007-09-21T22:38:00.000+08:00

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

12th Week of SIP- Microbiology

2007-09-16T12:48:00.000+08:00

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

11th Week of SIP- Clinical Chemistry

2007-09-11T22:50:00.000+08:00

Hi people!

Due to some miscommunition and confusion between the ever-busy medical technologists, this posting has been delayed. Sorry for the inconvenience caused!

Recap
Do u guys still remember what test did Adrian share about in his previous post? Yes, thats right! G6PD screening! (see week's 6 posting for the test in detail.)

After reading his post, you will wonder what happens after the patient has been screened G6PD-deficient (no fluorescence) of intermediate (having weak fluorescence). Since his workplace does not perform the next confirmatory step, he has to send it over to the lab I work in for G6PD quantitation. G6PD quantitation is the measurement of the ACTUAL G6PD enzyme level of the patient, while the initial screening only indicates "deficient", "intermediate" or "normal".

In other words, the initial screen is only QUALITATIVE, while the confirmatory test is QUANTITATIVE.

Today, I shall put an ending to the G6PD testing story by explaining the QUANTITATIVE aspect of G6PD testing.

Glucose-6-Phosphate Dehydrogenase (G6PD) Quantitation, Blood

Principle
The screening test for G6PD is based on the following immunofluorescence principle:

Glucose-6-Phosphate + NADP+ ----( G6PD)----> Gluconate-6-phosphate + NADPH (Fluorescence) + H+

G6PD (Glucose-6-Phosphate Dehydrogenase) catalyzes the oxidation of G-6-P to Gluconate-6-phosphate and simultaneously reduces NADP to NADPH. The formation of NADPH is monitored under 340nm. The rate of NADPH formation is proportional to the G6PD activity.

Procedures
Preparation of RBC haemolysate
1. Wash the EDTA blood 1 time with 0.9% saline.
2. Centrifuge to pack the cells at 3000rpm for 10 minutes.
3. Remove the saline and buffer coat completely.
4. Pipette 200 uL washed spun cells into 200 uL normal saline to obtain 1:1 RBC suspension. Mix well.
5. Pipette 50 uL of this 1:1 suspension into 250 uL of 1% saponin for lysis.
6. Mix well and stand for 2-80C for 10 minutes.
7. The haemolysate is ready for testing.

Aliquot the haemolysates onto autoanalyzer cups and load onto analyzer.

Clinical Significance
In addition to hemolytic anemia, G6PD deficient individuals can expect several other clinical manifestations of their condition. These include neonatal jaundice, abdominal and/or back pain, dizziness, headache, dyspnea (irregular breathing), and palpitations.

Treatment
Treatments for neonatal jaundice and hemolytic anemia have existed for many years. These treatments insure that the body tissues will be provided with enough oxygen by the red blood cells. Infants with prolonged neonatal jaundice are placed under special lights, called bili-lights, which alleviate the jaundice. When an anemic episode occurs, individuals are treated with nasal oxygen and are placed on bed rest, which may afford symptomatic relief. Anemic individuals are sometimes treated with human haptoglobin products, and/or blood transfusions. In acute hemolytic anemia, patients are administered folic acid.

Last Words

Thats all folks! All the best for ur MP! Remember to get plenty of rest and water! :)

Kent Lieow
TG01
0503261J

10th Week of SIP- Lab Techniques (Research)

2007-09-09T15:14:00.000+08:00

Hello all!
Hope you guys are enjoying work. Haha.

Anyway, today I'll be talking about the SOD Assay.

Superoxide Dismutase (SOD) Assay

What is SOD?
SOD is a metalloenzyme that catalyzes the dismutation of superoxide radicals (O2•-) into hydrogen peroxide (H2O2) and oxygen (O2).
Superoxide radicals are produced during metabolism in the mitochondria, muscle contraction, and as well as by NADPH oxidase during infections. Accumulation of these radicals would then lead to oxidative stress and eventually to diseases like Parkinson's disease, atherosclerosis etc.
Thus, with SOD present, it can defend against oxidative stress.

Other than SOD, Gluthathione peroxidase and Catalase also helps to protect against oxidative stress.

Liver supernatant will be used for this assay.

Principle of SOD Assay

The SOD Assay also uses tetrazolium salt to detect superoxide radicals. Xanthine oxidase present in the reaction mixture interacts with the radicals, which reduces the tetrazolium salt into formazan. The presence of SOD will reduce the amount of radicals, and in turn reduce the amount of formazan formed. Thus, increased levels of SOD, will lead to reduced levels of formazan.

After adding the respective reagents according to the protocol, abs is read using an ELISA plate reader at 450nm.

Fig. 1: Principle of SOD Assay

In other words: ↑SOD, ↓O2•-, ↓ formazan formed, ↓ Abs

That's it!
Take care guys~

Charmaine Tan
TG01

9th week of SIP - Immunology

2007-08-25T00:10:00.000+08:00

Hi everyone! Is my turn again! These few weeks I am attached to the Immunology department of my laboratory. Sound fun but practically nothing much to do as mainly the tests is done automatically. My lab is using a system called ADVIA Workcell whereby it employ a unique sample handling process that integrates the ADVIA Centaur Immunoassay System and the ADVIA 1650 Chemistry System to manage the patients' samples. In Immunology, the main machine we are using is the ADVIA Centaur Immunoassay System.

Taken from

http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1009257~a_langId~e_-111~a_productId~e_172958~a_storeId~e_10001.htm

The tests which are done in the Immunology Department are for infectious diseases (e.g. HIV, Hepatitis, HCV, etc.), fertility (e.g. Prolactin, Progesterone, Follicle Stimulating Hormone, etc.) Thyroid Function (e.g. Free T4, Free T3, TSH, etc.), and more (can't possibly name everything out.). Some are scheduled tests; some are tests that are run everyday. And of course, my department also does Dengue Testing. But I will more focus on the HIV testing using ADVIA Centaur Immunoassay System as the samples are the most in the department and it has it own Centaur machine (just for running HIV).

IMMUNOLOGY - HIV TESTING

Human Immunodeficiency Virus (HIV) is a retrovirus that can lead to Acquired Immunodeficiency Syndrome (AIDS), a condition whereby the human's immune system begins to fail, leading to life threatening opportunistic infections. HIV primary infects vital cells in the human immune system such as T-helper cells, macrophages and dendritic cells. It will lead to al low level of CD4+ cells number to decrease below critical through direct viral killing of infected cells, increase rate of apoptosis in infected cells and killing of infected cells by lymphocytes that recognizes the infected cells. This will cause the cell mediated immunity will lost and the body will increase susceptibility to infection. Infection can occur through several routes such as unprotected sexual intercourse, contaminated needles, transmission from infected mother to her baby at birth or through feeding breast milk. Treatment is now available but it will only prolong life span of the person but cannot fully cure the person.

The assay used for testing HIV is HIV 1/O/2 enhanced assay (EHIV). The samples we need to test for HIV is the patients' serum (preferably from plain or EDTA tube). This assay is an in vitro diagnostic immunoassay for qualitative determination of antibodies to HIV Type 1, Group O, and/or Type 2 in serum. The assay is an antigen bridging microparticle cheminluminescence immunoassay used for the detection of antibodies to the HIV in serum.

Principle of the Assay:

It is a two wash antigen sandwiched immunoassay in which antigen are bridge by the antibody present in the patient sample. The Solid Phase contains a preformed complex microparticles and the HIV antigen. This reagent is used to capture anti-HIV Type 1 and Type 2 recombinant antigen that is used to detect anti-HIV antibodies bound to the Solid Phase in the patient's sample.

Before Automation Steps are performed by the machine, some steps need to done manually:

Order the test by batch (by rack number). Note that each rack can only contain 5 sample tubes.
Remove the caps of the sample tubes and make sure the appropriate barcode is pasted on it and the volume of the serum is sufficient. If not, the serum need to be aliquot out into a new test-tube with the patient barcode pasted on it.
Place the sample tubes on the 1st rack ordered and place in the machine. Make sure the tubes sit in properly to prevent prop crashed.
Place the last few tubes on the last rack ordered. In between any racks can be used, meaning that the racks that are used in between the two racks ordered will be tested for HIV.
Then the machines will take in the racks one by one.

Automatic steps that are performed by the machine:

50ul of patient's serum will be aliquot out form the sample tube into a cuvette and incubate for 6 min at 37°C.
Reagents will be aliquot into the cuvette and incubate again for 18min at 37°C.
The cuvette will then wash with Wash 1.
Another reagent will then be aliquot into the cuvette and incubate for 18min at 37°C.
The cuvette will then wash with Wash 1 again.
Acid Reagent and Base Reagent will then aliquot into the cuvette to initiate cheminluminescence reaction.

Reference Range:

Reactive is more that 0.9
Non-reactive is less that 0.9

If Reactive:

Repeat the testing again. Spin down the sample tube at 10000rpm for 10 min, aliquot the serum out into a new test tube with appropriate barcode, then rerun in another machine.
If repeat test is negative, report as "Not Detected".
If repeat test is positive, call clinic to inform Doctor that Western Blot need to be done for confirmation.
If Western Blot is negative, attached the report to screening report and send directly to Doctor.
If Western Blot is positive, reports need to be notified to referring Doctor via phone and the report will be sent directly to the Doctor in a confidential envelope and CDC will be informed.
The reactive samples will be sent to hospital for Western Blot test.

No wonder Ye Tun, you tell me that you received samples from my lab. You all should know where the Western Blot is done by now.... Hope my post helps you all to understand HIV testing. Feel free to ask me questions.

Lizzie Chew, TG01, 0503194C

8th week of SIP - Flow Cytometry

2007-08-23T14:56:00.001+08:00

sorry for this late entry...

Flow Cytometry
Flow cytometry is a technology that simultaneously measures and analyses physical or chemical characteristics of single particles, usually cells, delivered in a flowing stream to the laser beam. The properties measured include the particle's relative size, granularity and fluorescence intensity. The flow cytometer I'm currently using is known as FACSCalibur from Becton Dickson.

Fig 1: FACSCalibur. Taken from http://igc-wiki.igc.gulbenkian.pt/lib/exe/fetch.php?w=&h=&cache=cache&media=uic:flowcytometry:facscalibur.jpg

Mechanism of FACSCalibur
The cytometer consists of 3 main subsystems: fluidics, optics and electronics. The fluidics systems is responsible for the transportation of cells, from 0.2-150µm in size, in a sheath stream to the observation point where the laser intercepts the cells (refer to Fig 2).. Only 1 cell should move through the laser beam at a given moment. To achieve this, the sample is injected into a stream of sheath fluid within the flow chamber (flow cell). The flow of sheath fluid accelerates the cells and restricts them to the center of the sample core, a process known as hydrodynamic focusing. Priming of the instrument to remove air bubbles and debris in the fluidics system is necessary to ensure proper interception of cells at the laser beam.

Fig 2: Inside the flow cell. Taken from http://users.path.ox.ac.uk/~nrust/diag1.gif

In the optics system (refer to Fig 5), the laser will illuminate the cells in the sample stream and light signals are generated, which in turn will be processed by the light detectors. Forward scattered light (FSC), light scattered just off the axis of the incident laser beam in the forward direction, is detected by a photodiode. A photodiode is a mechanical obscuration in the path of the laser beam that prevents the laser light itself from reaching the FSC detector, thereby allowing the detection of the FSC. The FSC signal is proportional to the cell-surface area/size (refer to Fig 3). Therefore, the larger the cells, the more intense is the FSC signal. Side scattered light (SSC) is collected at approximately 90° to the laser beam by a collection lens and then redirected by a beam splitter to the SSC detector (photomultiplier tube). It is proportional to the cellular granularity (refer to Fig 3). Correlated measurements of FSC and SSC are useful in distinguishing the different cell populations in blood sample (refer to Fig 4).

Fig 3: FSC and SSC. Taken from http://www.uams.edu/flowcytometry/Images/FSC&SSC.jpg

Fig 4: Cell popultions based on FSC vs SSC. Taken from http://www.flowlab.ucalgary.ca/flowlab/images/FSC.gif

Emission of fluorescence by the fluorochromes conjugated to a monoclonal antibody is also collected by the same lens that collects the SSC. The fluorescence signals are directed to their respective detectors via a system of mirrors and optical filters. The first mirror that collects light encounters is a 560SP (short pass) mirror, which allows light shorter than 560nm to pass and reflects light longer than that to other optical filters. The light that passes through this mirror then reach a beam splitter that reflects 10% of the light to SSC detector and lets the remaining 90% pass, which will reach the FL1 detector for short wavelength (green) fluorescence. A 530/30 bandpass filter is placed in front of FL1 detector to allow only light of wavelength 530 ± 30nm to reach the detector, eg FITC. The bandpass filter serves to optimise the specificity of the detector. Light reflected by 560SP mirror will reach a 640LP (long pass) dichroic, a beam splitter. Wavelengths shorter than 640nm are reflected to the FL2 detector for medium wavelength (yellow/orange) fluorescence, and longer wavelengths are passed and will eventually reach the FL3 detector through a 670LP filter. The amount of fluorescent signal detected is proportional to the number of fluorochrome molecules on the particle.

PS: Sorry guys.. it's kinda hard to digest at first, but hope u'll understand.. From Fig 5, you'll notice a half mirror placed just after the 640LP filter rite? dun risk asking me its purpose.. it's more confusing.. It just basically for FL4 detector.. wad's FL4 detector purpose? also pls dun take risk.. just pretend it's not there.. thanx..

Fig 5 (below): Optics System in FACSCalibur. Taken from http://www.sickkids.ca/fcf/images/FACSlayout.gif

Fig 6 (below): Examples of fluorochromes detected by different detectors. Taken from http://images.google.com.sg/url?q=http://facs.scripps.edu/images/facscolors.jpg&usg=AFQjCNGHeSQIyJ8hHvjT2spGEVX5e-ZWRg

All the signals detected are converted into electronic signals by the electronic system, and then processed by the computer. Analysis of the cell population can be displayed in several formats. The format I'm using is dot plot, which provides a two-parameter display of data. Each dot represents 1 or more events. Gating is required to restrict the analysis to only the cell population of interest. In Fig 7, the lymphocytes population is gated (in red colour) on the left graph, and therefore the resulting display on the right would only reflect the fluorescent properties of only the lymphocytes. A quadrant marker is used to divide the two parameter plots into four sections to distinguish populations that are considered -ve, single +ve or double +ve. The lower-left (LL) quadrant display events that are -ve for both parameters. The upper-left (UL) quadrant display events that are +ve for the y-axis parameter (CD4) and the lower-right (LR) quadrant are events that are +ve for the x-axis parameter (CD8). The upper-right (UR) quadrant display events that are positive for both parameters (CD4/CD8), or double +ve. Gated events are expressed in the form of percentage.

Fig 7: Dot plot with a gate emcompassing the lymphocyte population. Taken from http://www.bio.umass.edu/micro/immunology/facs542/hdots.gif

If you're still reading, thanx so much for ur time! distinction for ur patience! tried to minimise the information load.. but it still seems like... haix.. took a month to understand this flow cytometer.. I should say its inventor must be real free and nth else better to do.. but nevertheless, it's a veri much useful instrument anyway (ability to perform 10 000 events, shorter turnaround time).. Should you have any qn.. (erm.. can try not to ask?) ask simple qn will do.. and not too many pls.. thanx so much!

Joan
TG01

7th week of SIP - Virology

2007-08-13T21:44:00.000+08:00

Hi Everyone!

I am happy to be here to share my experience in SIP and some of the techniques/tests I learnt. Basically virology has 4 sections: Viral Serology lab; Viral Immunofluorescence (VIF) lab; Viral Isolation (VI) lab & Tissue Culture lab. *BSL2 lab.

As virology deals with lived viruses (especially viral isolation), potentially biohazardous materials and potentially dangerous chemicals (carcinogenic, cytotoxic, etc), safety procedures must be strictly followed. BSL2 safety cabinets are used and PPE are necessary.

I have gone to viral immunofluorescence and I am now in the tissue culture and viral isolation. I found that all the sections are related to each other, e.g. tissue culture grows cells for viral isolation. Immunofluoresecence is used as a confirmatory test for viral isolation and also used for mycoplasma testing of cell-lines.

::Medical Microbiology

I am sharing about flu surveillance which needs tissue culture, viral isolation and immunofluorescence techniques. Specifically I am sharing about flu surveillance shall-viral samples.

Clinical significance

When we receive the specimens for flu surveillance, we are screening for whether the sample has flu A or flu B or no flu virus. If the sample is found flu A positive, viral identification needs to be done to see whether it is H1, H3 or H5. If the sample is found H5, it can be H5N1(avian flu) and the further testing must be done in the Virology BSL-3 lab. Results are then recorded and reported to the public-health regulatory authorities.

Sample receiving/processing

Flu surveillance samples are usually respiratory samples like BAL (bronchial lavage), NPA (nasal pharyngeal aspirate), throat swab, lung autopsy specimens. They are received by viral isolation lab and the specimens are processed accordingly.

When receiving the sample, BAL, NPA and swabs should come in Hanks' transport media (prepared by tissue culture lab). Hanks' media is to let the virus survive before the specimens is processed and the viruses are isolated. I have observed the processing of BAL and swabs. BAL has to be centrifuged at 3500 rpm for 20 min at 4C. After that supernatant is used for inoculation into the cell culture tube. Swabs just need to be vortex and they are ready for inoculation.

Principles of the tests

Shell vial assay (viral isolation)

MDCK is the most sensitive cell lines for influenza A and B. MDCK cells are grown in the shell-vials. Shell-vials are the sterile yellow-cap 5ml tubes with flat bottom with a round sterile coverslip in each tube. MDCK cells grow on the surface of the coverslip. Then the shell vials are inoculated with processed specimens and they are centrifuged to allow the cells and viral antigens stick to the coverslip. After 48 hours of incubation at 33C, shell-vials are ready to be tested by indirect immunofluoresence assay (IFA).

NOTE: All the viral procedures are carried out in class2 biosafety cabinet.

Indirect immunofluoresence assay (IFA) for viral identification

Monoclonal antibodies specific to flu A and flu B are used to detect the presence of flu A or flu B antigens in the specimen. The principle is that if the viral antigens are present in the cells, monoclonal antibodies (MAb) specific to that antigen will bind to the antigens. Then secondary antibodies containing FITC (fluorescence substrate) conjugate will bind to monoclonal antibodies. When viewed under the fluorescence microscope, FITC substrate was exicted by UV light and give apple-green coloured fluorescence. Therefore a positive test is indicated by the presence of apple-green coloured fluorescence. Conjugate also contains a counter stain, Evan's Blue, which stains the cells dull red. Hence, positive test is seen as dull-red background with green fluorescence while negative test is seen as dull-red without any fluorescence.

Firstly cells grown in shall vials are scraped and cells are resuspend in phosphate buffer saline (PBS) just to get adequate concentration of cells to be spotted on the wells of the slide. The cell suspension is then spotted on the wells. Two wells (one for flu A and the other for flu B) will be used for each specimen. Then the slide is air dry. When the slide is dry, the slide is fixed in cold acetone for 10 mins, monoclonal antibodies (MAb) specific to flu A and flu B are added to respective wells and the slide is then put inside the humidity chamber and incubated at 37C for 30 minutes. After incubation, the slide is rinsed with PBS and then washed with PBS for 10 minutes (immerse in PBS and use the shaker). This is to remove excess and unbound monoclonal antibodies in the wells. Then the conjugate specific to flu A and flu B MAb is added to respective wells and the slide is then put inside the humidity chamber and incubated at 37C for 30 minutes. After incubation, the slide is rinsed with PBS and then washed with PBS for 10 minutes (immerse in PBS and use the shaker). This is to remove excess and unbound conjugate in the wells. After that, the slide is air dry, mount and observed under the fluorescence microscope.

If the specimen is flu A positive, the cell suspension of that specimen is again spotted on the three wells labelled H1, H3 & H5. The procedure is same as mention above, it's just that monoclonal antibodies specific to H1, H3 & H5 of flu A are used. H1 is reported as H1N1, H3 is H3N2 and H5 is sent to BSL3 lab for further testing.

I think I covered basic principles, procedures and significance of the tests. If you have any question, feel free to ask, regarding to viral isolation, tissue culture, immunofluorescence and other things. Actually viological tests (Viral Immunofluorescence (VIF); Viral Isolation (VI) & Tissue Culture) are quite time-consuming, labour-intensive and need special facilities and techniques and everything is done manually. Viral serology is 70% automated.

All the best for your SIP and MP!

Cheers

Ye Tun
TG01

6th week of SIP - Biochemistry

2007-08-04T22:51:00.000+08:00

Hey everyone,

This week, I was posted to the biochemistry lab. The lab consists of 3 sections.

1^st section

Consist of the main analyzer Beckman Coulter Synchron LX20 PRO Clinical System. Test for levels of Urea, creatinine, glucose, total protein, AST, GGT, etc. in blood serum.

2^nd section

Consist of analyzers to conduct tests such as

Blood gas
Serum lactate and ammonia test
Serum bilirubin
HbA1c to test for diabetes
G6PD testing
Serum and urine osmolality.

3^rd section

Also called the ‘urine bench’. Conduct tests such as

Urine FEME
Fungal test
Occult blood

Everyday, we receive the specimens, which comes together with a request form and a sticker label. We check the name and NRIC of the patient and the tests requested on the form. We then centrifuge the specimens for 10mins. However, some specimens for tests like HbA1c and Urine FEME does not require centrifugation. Blood specimens come in a plain tube (contain silicon gel), EDTA tube, or fluoride tube. After centrifugation, we will then process the specimens according to the tests requested.

I will explain more on G6PD testing.

G6PD Testing

Blood specimens come in EDTA tube to prevent clotting.

Prepare empty tubes for the samples and controls.

3 controls. Deficient control (commercial), intermediate control (commercial), and positive control (patient sample)

100µl of buffer (to lyse the cells) is aliquoted into each tube.

Aliquote 5µl of samples into the tubes containing the buffer.

Shake the tubes vigorously to mix, then incubate for 10mins to fully lyse the red cells, thus releasing the G6PD enzymes.

Prepare the cards to test for fluorescence. On each card, there are blank circles for each sample. Label the sample number and controls on the card, then proceed to aliquot 10µl of the sample solution and controls onto their appropriate circles.

Place the cards in an incubator for 10mins to dry the samples, as it will be difficult to see fluorescence if the samples are wet.

After incubation, place the cards in a UV illuminator, ‘Model CX-21 Ultraviolet Fluorescence Analysis Cabinet”.

The positive control should show fluorescence. Intermediate control should show slight fluorescence, and the deficient control should show NO fluorescence.

Check for fluorescence for the samples, to determine if the patient has G6PD deficiency. If they are deficient for G6PD, their sample will show no fluorescence, and these samples will be tested again just to confirm the results.

Theory behind G6PD Test

G6PD is an enzyme found in RBCs. G6PD deficiency is due to labile G6PD enzyme that is present in young cells but rapidly disappears with cell aging. It results in hemolysis after exposure to certain oxidant drugs such as anti-malarials, and also during infections. The disease is transmitted as an X-linked recessive, and is more prone in males.

Alright, that is all for now. Feel free to ask any question. Hope you guys are enjoying yourselves just like me! =D

Out.

Adrian Tan TG01
0503205G

5th week of SIP- Clinical Chemistry @ *GH

2007-07-28T00:00:00.000+08:00

Introduction (skip if necessary)

I'm attached to *GH Clinical Lab together with 4 others (names are omitted for confidentiality purpose). In *GH, there are 2 clinical labs. One 24hr routine lab situated in the main building for routine tests (basic chemistry, LFT, TFT, Renal function, etc), and another lab in a building that some lecturer in school has been warning us, where you gotta show your pass or risk "getting shot". Here, highly specialized tests such as specific proteins (rheumatoid factor), trace metal panel (copper, zinc) and others like G6PD, homocysteine, etc, are analyzed.

Our supervisor has kindly planned a schedule for us which indicates that for the span of 20 weeks, we will spend 10 in the 24hr routine lab, 5 purely on our MP (but still gotta report for work =/) and the last 5 at the "special" lab.

The routine lab is further divided into 5 stations, based on the type of tests performed and machines used. We will spend 2 weeks each at each station.

Background Information

This week, I'm attached to a station using the Beckman Coulter Unicel DXI (below)to conduct routine tests for serum ferritin, folate (RBC and serum), Vitamin B12 (usually done with folate as both are linked by the reaction pathway for methionine synthesis), Cortisol (24h urine free cortisol and serum cortisol), PTH, testosterone, D-HEAS (precursor of sex hormones) and beta-HcG (pregnancy hormone). The other analyzer, the Johnson & Johnson's Vitros DT60 II (image further down) tests for lipase, lactate, ammonia, lithium and cholinesterase.

Beckman Coulter Unicel DXI (taken form http://www.beckmancoulter.com/products/instrument/immunoassay/UniCel_DxI_800.asp)

Johnson & Johnson's Vitros DT60 II

Assay and Analyzer

For this posting, I will elaborate on the lactate assay of the Vitros DT60 II. (I chose the DT because its small compared to other analyzers but very, very unique.)

The beauty of the DT60 lies in the fact that unlike any other analyzer, you don't have to worry about adding or changing reagents..at all. As if that isn't enough, QC is done once a day (other analyzer needs at least two QC runs a day), and calibration is only done twice a year!

Other additional but rather significant benefits will be that minimal daily maintainence is necessary (just wipe with a cotton swab) and solid waste can be dumped of really easily.

Many of you will be wondering what kind of freak analyzer is this that does not need reagent loading. Well, that is because this machine conducts tests by using a special slide containing the required reagents! So you just have to load in that slide, drop a patient fluid sample onto it, and wait for the results. The slide will then be transported into the machine to carry out the test reactions.

The below shows the various layers of this multi-layered slide (click to enlarge).

(taken from http://www.orthoclinical.com/Products/products.aspx?id=4§ion=features)

The Vitros Slide is made up of five main layers. From the top, there is an upper slide mount (for supporting purposes). The next layer is the spreading layer, which helps to distribute the patient sample evenly to the underlying layers. The third layer is the reagent layer, which contains all the reagents needed for the photometric reaction to take place. The final layers are the transparent support layer (to aid photometric absorbance reading), and the lower slide mount (similar to the upper slide, for supporting purposes).

Test principle and reactions

Principle: Colorimetric Assay

Reaction:
In the case of the lactate assay, the reaction at the reagent layer of the dry-slide consists of two main reactions.

First, lactate is oxidized by lactate oxidase (found in the reagent layer) to pyruvate and hydrogen peroxide.

Second, the hydrogen peroxide generated oxidizes the 4-aminoantipyrine, 1, 7-dihydroxynapthalene dye system (included in the reagent layer) in a HRP (horseradish-peroxidase)-catalyzed reaction to form a dye complex (red).

The slide is incubated and the intensity of the dye complex is measured using the in-built spectrophotometer to quantitate the lactate values using its absorbance (at 555nm).

Notes:
This colorimetric assay has an approximate five minutes incubation time. When the test is complete, the results will be printed out immediately from the DT60 analyzer in the form of a narrow result slip. The results are written down on a form and filed into the LIS. The result slips are then torn off and clipped together with the result forms in a file.

Request form with attached heparinized tube in ice- Samples must be sent in ice (bag containing ice in water) to prevent anaerobic glycolysis from producing lactic acid!

Clinical Significance of Lactate test

Reference Range: 0.90 -1.70mmol/L

Elevated lactate levels indicates lactate acidosis. Lactate levels increase in many conditions and may aid the monitoring of conditions such as diabetes mellitus. In diabetes mellitus, renal failure may result, causing metabolic acidosis. Elevated lactate levels are also useful in diagnosis of tissue hypoxia, in which there is cellular glycolysis, hence causing respiratory acidosis.

Finally a raised lactate level may be used in the screening of malignancies.

Last Words

Hope you've "enjoyed" reading the post. Do feel free to ask questions. Thanks!

P.S. Answer turnaround time: About 24hrs (from the next working day).

Signing off!
Kent Lieow
TG01
0503261J

Lab Techniques (Research)

2007-07-22T22:26:00.000+08:00

Hi there! Hope you guys are enjoying your work~

Right, I'll be focusing on the experiment that I did for my MP rather than SIP. That's
because the SIP job scope is mainly setting and clearing up after the
students' practicals. ~~So that's why wemusn't abuse our position as students when we come back. It's all hard work being a TSO.~~

As most of you know, I'm working on the same project as Joan, just only looking at different components.

After 3 continuous weeks of gavaging the mice, it was time to sacrifice them. The mice were anaesthesized with Ketamine, a dissociative anaesthesia, and Pamlin, a sedative through Intraperitoneal (IP) injection.
How much to be injected was worked out by using this formula:
Dosage to be given = (Weight of mouse(kg) X Drug Dose)/Concentration

After they were all sedated, blood was collected by Cardiac Puncture and transferred to EDTA Tubes to prevent them from clotting.

The mice were then euthanized by cervical dislocation. The mouse was placed facing down, its neck was held down in place and the tail was pulled hard swiftly. This will then break the backbone of the mouse and sever the nerves connecting the backbone and the brain. This is one of many humane methods of euthanizing.

Diagram 1 shows the position on euthanizing the mouse using cervical dislocation.

The heart was checked to see if it was still beating before continuing on with dissection.
For dissection, a dissecting tray, a pair of forceps and scissors were prepared by swapping with 70% alcohol to disinfect the equipments. The mouse was placed onto the dissecting tray with its stomach facing up, a midline incision was made by using the forceps to grab the skin and the scissors to cut. The spleen was removed and placed into a tube containing 1X Phosphate Buffer Saline (PBS). The spleen is found at the left side of the mouse, at the posterior. The liver was removed and placed into 1X PBS with 0.16mg/ml of Heparin. The liver is directly below the lungs/ribcage.

Diagram 2 shows how the dissection was done and where the organs are found.

Thereafter, each mouse was placed into the biohazard bag and placed into the freezer for the storage of carcasses.

LDH Assay
First part:
The spleens were taken to perform the LDH Assay.
Working in a flowhood, each spleen was homogenized by scraping them against a sterile cell strainer into a suspension in 10ml of RPMI + 10%FBS medium.
Working in the dark, 5ml of Lymph M were pipetted into 12 15ml centrifuge tubes.
* Lymph M is a solution that works by density gradient. After centrifugation, different cell layers will be formed by their densities, including the lymphocyte layer.
5ml of the splenocyte suspensions were slowly aliquoted into the tubes using a glass pipette so as not to disturb the interface.
The tubes were then centrifued at 1500g, for 20 minutes at room temperature.
The yellowish lymphocyte layer was removed using a glass pipette into a new sterile 15ml centrifuge tube.

Diagram 3 shows the different layers after centrifugation.

The removing the lymphocyte layer, the lymphocytes were then washed 2 times by adding 10ml of medium and centrifuging at 800g, for 10 minutes at room temperature, decanting the medium then resuspending the cell pellet in another 10ml of medium.
After washing, 1ml of RMPI + 2%FBS medium was added into each tube and the cell pellets were resuspended.
Using a haemocytometer and tryphan blue, the lymphocytes were counted.
YAC-1 cells cultured were also counted using the above method.
Calculations were made to challenge the lymphocytes at different cell concentration, namely, 50X, 100X, and 150X.
A 96-well plate was used for the challenging of the cells.
From here onwards, RMPI + 2%FBS medium was used instead of RPMI + 10%FBS.
Blanks, High and low controls, effector controls and the test samples were done in triplicates.
The plate was then incubated in a 37oC incubator with 5% CO2 overnight.

Second part:
The next day, all the suspensions were pipetted into eppendorf tubes and centrifuged at 1500g for 5 minutes at 4oC to form the cell pellets.
Cell lysing solution was made, by aliquoting 100uL of Triton X into 10ml of RPMI + 2%FBS and vortexing the tube for Triton X to dissolve.
The high control supernatants were decanted and 200uL of the lysing solution was added to lyse the cells.
2 plates of 96-wells plates were used for the assay as each tube was done again in triplicates.
A 20X dilution was made to each tube by adding 95uL of DI water to 5uL of each cell supernatants into the new 96-wells plates.
The reaction mixutre was prepared by pipetting 1ml MilliQ water to the lyophilzate catalyst for 10 minutes and vortexing it after 10 minutes.
The dye was thawed in the water bath.
250ul catalyst was pipetted to 11.25ml of dye into 15ml centrifuge tube for 100 tests and the mixture was mixed by inverting up and down.
This reaction mixture was poured into a resevoir and 100uL of it was pipetted into each well using a multi-channel pipette.
The plates were then covered with aluminium foil and incubated for 20 minutes.
The stop buffer was prepared by pipetting 80ul DI water to 20ul 5M HCl to a dilution of 1N HCl
* Always add acid to water slowly!
After incubation, 50uL of stop buffer was pipetted into each well using a multi-channel pipette to stop the reaction.
Absorbance were then read at 490nm by using a spectrophotometer.
*Any bubbles formed will affect the results, thus, all bubbles must be removed before reading the plate.
Cytotoxicity was calculated using this formula:
((Effector-Target Mixture – Effector Control) – Low Control))/(High Control – Low Control)

Diagram 4 shows YAC-1 cells at 20X

Diagram 5 shows the lymphocytes at 10X

Diagram 6 shows the lymphocytes with YAC-1 cells at 10X

All diagrams and photos taken/done by me.

That's all!
Any questions, just ask me! =DD

Posted by:
Charmaine

Lab Techniques (Research)

2007-07-18T19:36:00.000+08:00

FYI: I m attached to research lab under the Cellular and Molecular Research division. My lab focuses on gene structure and expression.

I was assigned to do a project to prevent 2 proteins from interacting to prevent tumorigenesis.
Throughout my SIP, I did different things everyday unless I don’t get any expected results and need to redo. My work rotates around Mbio, CellBio, MCT and Biochemistry. I am going to focus on DNA preparation in my discussion here. I am supposed to isolate the DNA I clone from the host cells that I had transformed.

There are three types of DNA prep for plasmid isolation: maxi-, mini, midi-prep.

Below are differences between these 3 prep works:

As I only did the maxi- and miniprep, I cannot comment more on midiprep.

Maxiprep:
-large scale DNA preparation
-Quantity of DNA eluted: 500ug/ml- 850ug/ml
Steps and reagents involved:
-Uses cultured cell clones in 250ml of LB media
- Requires centrifugation at 4۫C
- Requires a big Maxi- column to bind DNA, a dish to collect flow through that pass the column for disposal and a Du-pont tube for DNA elution
- Steps are lengthy and more buffers are involved like W8 (wash buffer), E4 (elution buffer), 70% ethanol for precipitation, TE buffer for resuspension and Equilibration Buffer (EQ1) to wet the column before draining any solution
- Drain through column by gravity flow
- Principle is lysis, bind and wash DNA, precipitation and elution.Details of the protocol can be found in www.invitrogen.com under PureLink™ HiPure Plasmid Filter Purification
Miniprep
-Small scale DNA preparation
-Quantity of DNA: up to 40ug/ml
-Steps and reagent involved:
-Uses cultured cells in 4ml of LB media
-Only requires a normal centrifuge
- Only requires a normal spin column to bind DNA and 2ml Wash tube to elute the DNA and for flow through collection
-Involves lesser steps and buffer and column work by centrifugation to bring the solution down.
- Principle works by alkaline lysis of the host cells, bind, wash and elution purified DNA.
- Details of the protocol can be found in www.invitrogen.com under PureLink™ Quick Plasmid Miniprep Kit
Midiprep
-Middle scale DNA preparation
-Quantity of DNA: 100-350ug/ml

After the prep for our DNA purification, I need to do a quantitation of DNA content using a spectrophotometer to check is there sufficient DNA for the following steps of the project. Quality of DNA is also monitored. DNA with ratio measured at 1.6-2.0 are considered pure and good quality. Sometimes, ratio may not be quantitated by the spectrophotometer if the concentration is too low. The Miniprep is done before maxi prep so that we can prepare our large amount of DNA with the correct sequence based on the sequence results of the clone from miniprep. We quantitate the DNA in miniprep for sequencing reaction to monitor if the DNA cloned has the right sequence so that we can proceed on our project. 1 ug/ml is sufficient.
For maxiprep, we check the concentration of DNA to see if there are enough for in vitro translation for translating DNA to proteins in the next part of project .
Running a gel with the same aliquot of DNA can confirm the quality of DNA based on comparison of the intensity of the bands to make sure the measurement from the spectrophotometer is accurate. DNA might be contaminated with RNA, proteins and carbohydrates which can be determined by the quality ratio of the DNA and confirmed again using the gel.

I hope u all understand what I said and dont hesitate to ask. Thanks.

Ai Tee
0503160D
TG01

Haematology / Blood Banking

2007-07-07T21:36:00.000+08:00

Actually I am attached to Quest Lab, within the lab, we, as an intern, will rotate to different department (Hematology, Biochemistry, Immunology, Serology, Microbiology, Specimen Reception, Cytology/Histology and Paragon Lab) every three week. For these two week, I am attached to the Hematology department.

In the Hematology department, for the first week, my main duty is to do ABO blood group and Rh typing using plating method. For the second week, my main duty is to use CELL-DYN 3500/3700 to run full blood count test and malaria parasite test.

ABO blood group and Rh typing

Principle:

Label the plate as below.
------A---B---AB---D---α---β---O
1
2
3
4
5
6
7
8
9
10
11
12
Aliquot one drop of anti-A, anti-B, anti-AB and anti-D to the plate labeling A, B, AB and D respectively.
Spin down the patient blood sample to separate the red cells and plasma.
Aliquot one drop of plasma to the α, β, O column each.
Dilute the red cells in saline (estimate 3-5%).
Aliquot one drop of the diluted red cells to the A, B, AB, D column.
Aliquot one drop of commercial A, B, O cells too the serum.
Mix the blood with a vibrator and centrifuge it at 900rpm for 1 min 30 sec.
Read the result manually using a stick to mix to check for agglutination.

Test Result with reference range:

4>agglutination>positive
0>no agglutination>negative

Things to take note:

All reagent used are diluted. 1 drop of anti-A, anti-B, anti-AB, anti-D is diluted with 3 drops of saline. And one drop of commercial A, B, O cell to one drop of saline.
If Rh negative, Du testing is needed. Add one drop of diluted patient red cells to one drop of concentrated Anti-D in a new test tube. Centrifuge. Incubate at 37°C for 15 min. Wash with saline for 3X. Add one drop of AHG and centrifuge to check for agglutination.

CELL-DYN 3500/3700 to run full blood count test and malaria parasite test

CELL-DYN3500/3700 is normally given the red cell indices, platelets, WOC (WBC optical count), WIC (WBC Impedance Count) count.

Run internal QC and control:

Do background clean for 3-5 times to make sure the values stated on the screen is near to 0 (Stated on the PC screen connected to the machine).
Run 3 control samples (Low, Normal, High) using open sample mode (manual). Make sure all values are within control limit.
Run 2 Internal QC (normally is patient sample that is tested a day before and is within control limit) using closed and open mode. Make sure all values are within control limit.

Principle:

Load the sample into the machine for it to run in closed sample mode, and then wait for result.
For sampling error, redo the test using open sample mode.
Check whether all the values of the result are within control limit.
Mainly is to check for WOC, Hb, MCHC and platelets values.

Test Result with reference range:

For malaria parasite (MP), require making a smear when the platelets are <150>
For full blood count (FBC), to do a rerun, WOC - <2.0>25, Hb - <60>180, MCHC - <300>365 and platelets - <100> 750. Require to make a blood smear when WOC - <3>15 and platelets <150>150.

Clinical Interpretation:

MP is test whether the patient (normally foreign worker) have malaria parasites.
FBC is to test for any abnormal RBC, WBC and Platelets which is closely related to blood diseases such as leukemia and anemia.
MP cells normally found in patients: Plasmodium Falciparum

Taken from http://deep6inc.com/previewher20.html

Things to take note:

Controls and Internal QC need to be run at least 2 times a day to make sure the machines are accurate. This is to make sure that the test result for the patients sample run is reliable and accurate.
Closed sample mode is automated method, just need to load the sample for the machine to run. Therefore, the volume of the patient blood sample must be more than 1ml.
When using open sample mode, normally use for small amount or little sample, because is a manual method, make sure the patient sample is mix properly.

Hope you all can learn something from my post. Feel free to ask any question.

Lizzie Chew, TG01

Lab Techniques (Research)

2007-07-01T19:06:00.000+08:00

Posted by: Joanne

This is a summary of my first week attachment in school. During the 1st week, there was no SIP involved due to delay in assigning TSO to us. So we basically focus on our MP. My MP is on the effect of functional food on the murine B & T cells.

One week before SIP commence, the 20 mice models arrived. They were to be isolated in the quarantine room, away from the other animals in the animal holding unit (AHU). This is to prevent the spread of potential infectious disease to the healthy animals. Everyday, we had to go back to school to check on the mice health and update the daily environmental record. We also had to ensure that there was sufficient food and water supplied to the mice. As we were dealing with live animals, it is necessary to check on them everyday i.e Mon to Sun. Sometimes on weekends, we had to wake up early just to go school and do a 15min check on them. It was kind of tiring at first but we have 5mths to let us get used to it.

First week of SIP - We prepared the solutions for the negative/postive control group and test group. Autoclaved water was used as a negative control with basal production of lymphocytes. Vitamin C was used as a positive control which will enhanced the production of lymphocytes. The method used to adminster the article/solution was by gavaging. Gavaging refers to the force feeding of article directly into the stomach with the insertion of gavage tubes from the mouth. This process was to ensure that each mouse recieved the specific amount of article administerd to prevent any potential pre-analytical variations. It had to be done carefully to avoid inappopriate insertion into the lungs instead. One of the identification method we used was cage cards for each group. It indicates the protocol number, strain, sex, age, supplier, investigator and contact person. We had done ear punching to ease the identification of every mouse. It involve the use of a special punch to produce a small notch near the edge or in the middle of the ear.

The above content was what I'd done for this week. If u have any questions, pls feel free to ask. Take care!

Taken from: http://www.jhu.edu/animalcare/images/rat_james_mouse_identification.gif

MMIC: Case Study 1

2007-05-05T16:06:00.000+08:00

Description of Potential Diseases:

Cat-scratch Disease (A,B)
Transmission vehicle: Most commonly by kittens, also spread by cats & dogs, with fleas as intermediate hosts
Bacteria: Bartonella henselae
Causes: Bites and scratches by infected vehicle, contact with infected vehicle’s saliva on open lesions
Signs and symptoms: Bump or blister at site of injury, swelling of lymph nodes near sit of injury, fatigue, low fever, headache, malaise & enlarged spleen (less common)
Epidemiology: Found world wide (1, 9)

Lyme Disease (A,B)
Transmission vehicle: Ticks on cats and dogs.
Bacteria: Borrelia burgdorferi
Causes: Bite of infected tick.
Signs and symptoms: Fever, skin rash, joint inflammation, and flu-like symptoms
Epidemiology: USA (6, 10)

Rat-bite Fever (A,B)
Transmission vehicle: Fluids in the mouth or nose or urine of an infected rodent.
Bacteria: Streptobacillus moniliformis & Spirillum minus
Causes: Bite of an infected rodent or contact with its secretions
Signs and symptoms: Fever, rash (red or purple plaques), headache and muscle ache.
Epidemiology: Japan, Australia, Africa, North and South America, and Europe. (5, 12)

Rocky Mountain Spotted Fever (A,B)
Transmission vehicle: Ticks on cats and dogs.
Bacteria: Rickettsia rickettsii
Causes: Bite of infected tick
Signs and symptoms: Fever, rash, muscle pain, nausea, vomiting and diarrhea.
Epidemiology: Asia and USA (3, 9)

Toxoplasmosis (A, B)
Transmission vehicle: Most commonly in cats, and also by birds and other animals
Bacteria: Toxoplasma gondii
Causes: Ingestion of contaminated soil, careless handling of cat litter, ingestion of raw or undercooked meat
Signs and symptoms: swollen lymph glands, muscle aches, reduced/blurred vision, fever & headache
Epidemiology: More common in North America (2, 13)

Typhoid Disease (A,B)
Transmission vehicle: Ticks carrying the bacteria previously in contact with chronic stool carrier.
Bacteria: Salmonella typhi & Salmonella paratyphi
Causes: Ingestion of contaminated food or water, sewage & shellfish
Signs and symptoms: Severe headache, fever, loss of appetite, fatigue, constipation, diarrhea, abdominal tenderness, rose spots on lower chest and abdomen, high fever & bloody stools
Epidemiology: Found worldwide, especially in Asia, Africa, Latin America, the Caribbean, and Oceania (4, 7)

Typhus (A,B)
Transmission vehicle: Ticks and fleas on rats and cats.
Bacteria: Rickettsia typhi (murine or endemic) & Rickettsia prowazekii (epidemic)
Causes: Bite of infected flea or tick.
Signs and symptoms: Extremely high fever, dull red rash that begins on middle of the body and spreads, nausea, vomiting and headache
Epidemiology: USA (11, 14)

-------------------------------------------------------
Preliminary diagnosis:

The symptoms of these disease includes rashes, high fever, and general weakness (fatigue) (3, 4, 5, 6, 7, 8, 10, 11, 12, 14):
1. Lyme Disease
2. Rat-bite Fever
3. Rocky Mountain Spotted Fever
4. Typhoid Fever
5. Typhus

Cat Scratch Disease is rejected due to the following reasons:
- The symptoms of Cat Scratch Disease does not include rash, which is one of the prominent features of this situation
- The fever caused by Cat Scratch Disease is low, however, in this situation, the patient's fever is high. (1, 9)

Toxoplasmosis is rejected due to the following reason:
- The symptoms of Toxoplasmosis does not include both general weakness (fatigue) and rash, which are symptoms found on the patient. (2, 13)

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

References:

1. American Uveitis Society. (2003). Cat-Scratch Disease. Retrieved May 5, 2007, from http://www.uveitissociety.org/pages/index.html > Search> Search: Cat scratch disease > Cat-Scratch Disease

2. Centers for Disease Control and Prevention. (2004). Toxoplasmosis. Retrieved April 25, 2007, from http://www.cdc.gov/index.htm > Search: Toxoplasmosis > Toxoplasmosis: Index | CDC Parasitic Diseases > Fact Sheet: Toxoplasmosis

3. Chinese University of Hong Kong. (2006). Rickettsia. Retrieved May 6, 2007, from www.aic.cuhk.edu.hk > Search Rickettsia > Rickettsia

4. eMedicine. (2006). Typhoid Fever. Retrieved May 9, 2007, from http://www.emedicine.com/ > eMedicine Specialties > Medicine, Ob/Gyn, Psychiatry, and Surgery > Infectious Diseases > Typhoid Fever.

5. HeathAtoZ.(2002). Rat-bite Fever. Retrieved 10 May, from, www.healthatoz.com > Search: Rat-bite Fever > Rat-bite Fever

6. Mayoclinic.com. (2006). Lyme Disease. Retrieved May 9, 2007, from www.mayoclinic.com > Search: Lyme Disease > Lyme Disease

7. Medline Plus. (2005). Typhoid Fever. Retrieved May 9, 2007, from www.medlineplus.gov > Medical Encyclopedia > To-Tz > Typhoid Fever

8. Medline Plus. (2005). Rocky Mountain Spotted Fever. Retrieved May 9, 2007, from www.medlineplus.gov > Search: Rocky Mountain Spotted Fever > Rocky Mountain Spotted Fever

9. Medline Plus. (2005). Cat scratch disease. Retrieved April 25, 2007, from http://medlineplus.gov/ > Medical Encyclopedia > C-Cg > Cat scratch disease

10. Medline Plus. (2005). Lyme Disease. Retrieved May 8, 2007, from www.medlineplus.gov > Search: Lyme Disease > Lyme Disease

11. Medline Plus. (2005). Typhus. Retrieved May 7, 2007, from www.medlineplus.gov > Search: Typhus > Typhus

12. Medline Plus. (2005). Rat-bite Fever. Retrieved May 6, 2007, from www.medlineplus.gov > Search: Rat-bite Fever > Rat-bite Fever

13. Medline Plus. (2006). Toxoplasmosis. Retrieved May 5, 2007, from www.medlineplus.gov > Medical Encyclopedia > To-Tz > Toxoplasmosis

14. Wikipedia. (2007). Typhus. Retrieved May 10, 2007, from www.wikipedia.org > Search: Typhus > Typhus

*NOTE*
Direct links under (A,B)

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

Charmaine~
Lizzie~
Adrian~
Joan~
Ye Tun~
Ai Tee~
Kent~

MMIC: Case Study 1

2007-04-25T10:22:00.000+08:00

A 85-year-old male was found to be staying alone in a one-room flat. It is found that his mattress was soiled with human excreta and urine. The premise is infested with cockroaches, fleas and rats. It is known to the nieghbours that the elderly man has the habit of feeding stray dogs and cats in the neighbourhood.

The elderly man was referred to a nursing home and is presented with high fever, rash and general weakness.

How would you approach this situation in order to provide the final diagonosis of the suspected microorganisms?

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

Potential diseases:

1. Cat Scratch Disease
Medline Plus. (2005). Cat scratch disease. Retrieved April 25, 2007, from http://medlineplus.gov/ > Medical Encyclopedia > C-Cg > Cat scratch disease

Information on the definition, causes, incidence, risk factors, symptoms, signs and tests, treatment of Cat Scratch Disease.

2. Lyme Disease
Mayo Clinic. (2007). Lyme Disease. Retrieved April 25, 2007, from http://www.mayoclinic.com/ > Search: Lyme Disease > Lyme Disease

Information on the introduction, signs & symptoms, causes, risk factors, when to seek medical help, screening and diagnosis, treatment and prevention of Lyme Disease

3. Rat-bite Fever
Medline Plus. (2005). Rat-bite Fever. Retrieved May 7, 2007, from www.medlineplus.gov > Medical Encyclopedia > R > Rat-bite Fever

Information on the definition, causes, incidence, risk factors, symptoms, signs and tests, treatment of rat-bite fever.

4. Rocky Mountain Spotted Fever
Medline Plus. (2005). Rocky Mountain spotted fever. Retrieved April 25, 2007, from http://medlineplus.gov/ > Medical Encyclopedia > R > Rocky Mountain spotted fever

Information on the definition, causes, incidence, risk factors, symptoms, signs and tests, treatment of Rocky Mountain Spotted Fever.

5. Toxoplasmosis
Centers for Disease Control and Prevention. (2004). Toxoplasmosis. Retrieved April 25, 2007, from http://www.cdc.gov/index.htm > Search: Toxoplasmosis > Toxoplasmosis: Index | CDC Parasitic Diseases > Fact Sheet: Toxoplasmosis

Information on the definition, incidences, symptoms, who are the people at risk, what to do if at risk of severe toxoplasmosis or contracting toxoplasmosis, prevention, treatment, will infected cats be able to remain at home and will they always spread the disease to people.

6. Typhoid Fever
Medline Plus. (2005). Typhoid Fever. Retrieved May 9, 2007, from www.medlineplus.gov > Medical Encyclopedia > To-Tz > Typhoid Fever

Information on the definition, causes, incidence, risk factors, symptoms, signs and tests, treatment of typhoid fever.

7. Typhus
Medline Plus. (2005). Typhus. Retrieved May 8, 2007, from www.medlineplus.gov > Medical Encyclopedia > To-Tz > Typhus

Information on the definition, causes, incidence, risk factors, symptoms, signs and tests, treatment of typhus.

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

Charmaine~
Lizzie~
Adrian~
Joan~
Ye Tun~
Ai Tee~
Kent~

Group Expectations

2007-04-25T09:58:00.000+08:00

This is our final mile in the race. So in order to lead the pack, we have to work doubly hard in all our modules, be diligent and FOCUS! It's not all about memorising every single piece of article, but we too, have to understand our work. By understanding, we can then apply what we learnt in our future careers. So let's all pull up our socks and attitude!

~OUR GOALS~
Full participation
Punctuality
Optimistic
Flexibility
Responsible
Work Smarter

Group Members:
Charmaine
Ai Tee
Joan
Lizzie
Adrian
Ye Tun
Kent

GAMBATTE KUDASAI MINNA! XD
Charmaine~