The Royal Intro

MMIC PBL2 - Blog 2

2008-01-27T10:40:00.001-08:00

Fungal

Fungal Diseases	Treatment	Prevention
Tinea capitis(scalp ringworm)	-Oral antifungal medicines-Oral antifungal pills such as griseofulvin, terbinafine, itraconazole, fluconazole and ketoconazole	-Medicated shampoo maybe used to reduce the risk of spreading the scalp ringworm to someone else:such as selenium sulfide shampoo and ketoconazole shampoo -Keep skin dry and cool
Tinea cruris(jock itch)	-Antifungal creams or ills	-Keep skin dry and cool
Tinea pedis(athelete’s foot)	Antifungal ointment, lotion, powder or spray such as terbinafine (Lamisil AT), clotrimazole (Lotrimin AF) and miconazole (Micatin)- Oral medications such as itraconazole (Sporanox), fluconazole (Diflucan) and terbinafine (Lamisil)	-Keep skin dry and cool
Sporotrichosis	-Oral potassium iodide such as itraconazole (Sporanox)	nil
Histoplasmosis	-Antifungal medications such as amphotericin B (Fungizone IV) and itraconazole (Sporanox)	-No vaccine is available. -Itraconazole is used for chronic suppression,AIDS patients.
Disseminated candidiasis	- Antifungal medications such as Amphotericin B and Fluconazole.	-Predisposing factors shold be reduced or eliminated. -Oral thrush can be prevented by using Clotrimazole troches/nystatin "swish & swallow". -No vaccine is available..
Chronic mucocutaneous candidiasis	-Antifungal agents, immunologic therapies, and combination therapy -Such as ketoconazole	-Predisposing factors shold be reduced or eliminated. -Oral thrush can be prevented by using Clotrimazole troches/nystatin "swish & swallow". -No vaccine is available.
Cryptococcal meningitis	-Antifungal medications such as amphotericin B	-Fluconazole (Diflucan) is used to prevent the cryptococcal infection from coming back (maintenance treatment). -No vaccine is available.
Aspergillosis	-Oral corticosteroids-Antifungal medications such as amphotericin B and oriconazole	-No vaccine is available.

Virus

Dieases/virus	Treatment	Prevention
Hep A	There is no specific treatment for HAV and most people fight off the virus naturally, returning to full health within a couple of months. The doctor will advise avoiding alcohol and fatty foods as these can be hard for the liver to process and may exacerbate the inflammation.	Hepatitis A immunisation is given in a series of injections. The first single injection in the arm gives protection for a year. The second booster injection at 6 to 12 months extends protection for up to 10 years.
HEPATITIS B	In the majority of patients with active HBV, symptoms will not be severe and treatment will not be required Antiviral medication is given as treatment to those with chronic symptoms to help prevent further liver damage. These medications may be injected or given in pill form. Examples are Interferon Alpha, Lamivudine and Baraclude	Three immunisation injections are given over a period of 3-6 months. A blood test is taken once the course of injections is completed to check that they have worked. Immunity should last for at least 5 years.
NAIROVIRUS	The antiviral drug ribavirin has been used in treatment of established CCHF infection with apparent benefit. Both oral and intravenous formulations seem to be effective.	Avoidance of areas where tick vectors are abundant and when they are active (Spring to Fall); Regular examination of clothing and skin for ticks, and their removalUse of repellents.
WEST NILE FEVER	There is no specific treatment for West Nile virus infection. Intensive supportive therapy is directed toward the complications of brain infections. Anti-inflammatory medications, intravenous fluids, and intensive medical monitoring may be required in severe cases	When you are outdoors, use insect repellent containing an EPA-registered active ingredient.
RABIES	If rabies vaccine treatment is called for, it should be started as soon as possible after exposure. Counting the first day of vaccine treatment as day 0, injections are administered on days 0, 3, 7, 14, and 28.	The rabies vaccine is administered after exposure to the virus. No matter where the wound, authorities emphasize that the first and most valuable preventive measure is thorough cleaning of the site with soap and water, and immediate medical attention.
JAPANESE ENCEPHALITIS	nil	In general, vaccine should be offered to persons spending a month or longer in endemic areas during the trans-mission season, especially if travel will include rural areas. Use of insect repellants
YELLOW FEVER	Serious cases of yellow fever always need hospital treatment. As there are no products that combat the virus itself, the doctor can only treat the symptoms. If there is a lack of fluid in the body, leading to disturbances in the electrolyte balance, this can be remedied by administration of fluids by intravenous drip. In mild cases, the pain may be relieved with simple painkillers. High temperatures can be treated by cooling the patient and giving them appropriate medicines to lower the temperature, such as aspirin (eg Disprin) or ibuprofen (eg Nurofen).	Vaccination available: This vaccine contains a live, weakened form of the yellow fever virus. It provokes the body's immune response without causing the disease.
Leishmania virus	itraconazole/ Fluconazole/ Paromomycin/ miltefosine (impavido)	· Stay in well-screened or air-conditioned areas as much as possible. Avoid outdoor activities, especially from dusk to dawn, when sand flies are the most active.· When outside, wear long-sleeved shirts, long pants, and socks. Tuck your shirt into your pants. · Apply insect repellent on uncovered skin and under the ends of sleeves and pant legs. Follow the instructions on the label of the repellent. The most effective repellents are those that contain the chemical DEET (N,N-diethylmetatoluamide). The concentration of DEET varies among repellents. Repellents with DEET concentrations of 30%-35% are quite effective, and the effect should last about 4 hours. Lower concentrations should be used for children (no more than 10% DEET). Repellents with DEET should be used sparingly on children from 2 to 6 years old and not at all on children less than 2 years old.· Spray clothing with permethrin-containing insecticides. The insecticide should be reapplied after every five washings. · Spray living and sleeping areas with an insecticide to kill insects.· If you are not sleeping in an area that is well screened or air-conditioned, use a bed net and tuck it under your mattress. If possible, use a bed net that has been soaked in or sprayed with permethrin. The permethrin will be effective for several months if the bed net is not washed. Keep in mind that sand flies are much smaller than mosquitoes and therefore can get through smaller holes. Fine-mesh netting (at least 18 holes to the inch; some sources say even finer) is needed for an effective barrier against sand flies. This is particularly important if the bed net has not been treated with permethrin. However, it may be uncomfortable to sleep under such a closely woven bed net when it is hot.NOTE: Bed nets, repellents containing DEET, and permethrin should be purchased before traveling and can be found in hardware, camping, and military surplus stores
RIFT VALLEY FEVER	Ribavirin	. Hepatotoxic medication as well as aspirin and NSAIDs should be avoided during the acute disease. Contact with sick or dead animals must be avoided.Smithburn vaccine (single dose, life-long protection), or vaccination with the formol-inactivated vaccine (boosters needed). For personal protection covering clothing (long sleeves, long trousers), insect repellents (best with DEET) and impregnated mosquito nets are adequate in normal situations. Barrier-nursing is indicated in the care of patients. There is still no commercial vaccine available for humans. It is true that experimental cell-culture vaccines for medical use do exist, but they are not easy to obtain.

General precautions to be taken when at camp
Preferably apply insect repellent whenever the individual is in the jungle and also apply before sleeping. This is to lower the risk of getting beaten by insects (sandflies, mosquitoes, etc.). Before consuming food, ensure to wash hands with clean water or boiled river/ stream water to reduce chances of consuming vectors of viruses. Ensure consumption of fluids from clean bottled water or boiled water from water bodies. Observe good personal hygiene to reduce chances of being infected by viruses and to reduce chances of transmitting viruses to other platoon members. Quarantine sick individuals to reduce chances of spreading the viruses contracted. Clean wounds or cuts properly to reduce chances of infection.

Protozoa

Dieases	Treatment	Prevention
Schistosomiasis	Praziquantel/ Oxamniquine/ Mirazid	· Swimming in the ocean and in chlorinated swimming pools is generally thought to be safe · Drink safe water(don’t drink water coming directly from canals, lakes, rivers, streams or springs is safe, you should either boil water for 1 minute or filter water before drinking it. Boiling water for at least 1 minute will kill any harmful parasites, bacteria, or viruses present. Iodine treatment alone WILL NOT GUARANTEE that water is safe and free of all parasites. · Bath water should be heated for 5 minutes at 150o F. Water held in a storage tank for at least 48 hours should be safe for showering · Vigorous towel drying after an accidental, very brief water exposure may help to prevent the Schistosoma parasite from penetrating the skin. You should NOT rely on vigorous towel drying to prevent schistosomiasis Castor oil as an oral- penetration
Balantidiasis	Tetracycline/ metronidazoleiodoquinol / paromomycin.	· Purification of drinking water. Water can be purified by filtering, boiling, or treatment with iodine. · Proper food handling. Measures include protecting food from contamination by flies, cooking food properly, washing one's hands after using the bathroom and before cooking or eating, and avoiding foods that cannot be cooked or peeled when traveling in countries with high rates of balantidiasis. · Careful disposal of human feces. Monitoring the contacts of balantidiasis patients. The stools of family members and sexual partners of infected persons should be tested for the presence of cysts or trophozoites.
Cholera	Tetracycline/ azithromycin/ oral rehydration solution/ mixture or salts, sugar and water drunk in large amount	· Wash your hands. Frequent hand washing is the best way to control cholera infection. Wash your hands thoroughly with hot, soapy water, especially before eating or preparing food, after using the toilet, and when you return from public places. Carry an alcohol-based hand sanitizer for times when water isn't available. · Avoid untreated water. Contaminated drinking water is the most common source of cholera infection. For that reason, drink only bottled water or water you've boiled or disinfected yourself. Hot beverages such as coffee and tea as well as bottled or canned soft drinks, and wine and beer are generally safe. Carefully wipe the outside of all bottles and cans before you open them and ask for drinks without ice. Use bottled water to brush your teeth. · Eat food that's completely cooked and hot. Choose food that's been thoroughly cooked and is served hot. Cholera bacteria can survive on room temperature food for up to five days and aren't destroyed by freezing. It's best to avoid street vendor food, but if you do buy it, make sure your meal is cooked in your presence and served hot. · Avoid sushi. Don't eat raw or improperly cooked fish and seafood of any kind. · Be careful with fruits and vegetables. When you're traveling, make sure that all fruits and vegetables that you eat are cooked or have thick skins that you peel yourself. Avoid lettuce in particular because it may have been rinsed in contaminated water. · Be wary of dairy foods. Avoid ice cream, which is often contaminated, and unpasteurized milk. Cholera vaccine. Because travelers have a low risk of contracting cholera and because the traditional injected vaccine offers minimal protection, no cholera vaccine is currently available in the United States. A few countries offer two oral vaccines that may provide longer and better immunity than the older versions did. If you'd like more information about these vaccines, contact your doctor or local office of public health. Keep in mind that no country requires immunization against cholera as a condition for entry.
Amebiasis	metronidazole, paromomycin, iodoquinol, or diloxanide furoate	· Drinking only water that has been bottled in sanitary conditions or boiled (water-purifying tablets are ineffective against amebic cysts) · Eating only cooked or peeled vegetables or fruits · Protecting food from fly contamination Washing hands after defecation and before preparing or eating food
Cryptosporidiosis	Nitazoxanide	Stools of patients with cryptosporidiosis are highly infectious · Frequent washing of the hands, including under the fingernails · Not eating unwashed fruits unless peeled · Boiling water if there is any doubt about its source · Boiling stream or river water for three minutes · Using filter paper with pore size of 1 micro<>
Cyclosporiasis	trimethoprium- sulfamethoxazole/ bactrim / septra/ cotrim	· Drinking only water that has been bottled in sanitary conditions or boiled (water-purifying tablets are ineffective against amebic cysts) · Eating only cooked or peeled vegetables or fruits · Protecting food from cyclosporasis contamination Washing hands after defecation and before preparing or eating food
Toxoplasmosis	pyrimethamine / trisulfapyrimidines / sulfadiazine./ folinic acid/ spiramycin	· Make sure your physician checks your blood for toxoplasma antibodies . Use work gloves and wash your hands afterwards. · Control flies and cockroaches as much as possible. They can spread contaminated soil or cat feces onto food. · Avoid eating raw or undercooked meat (or poultry) and unwashed fruits and vegetables. · Wash your hands thoroughly before you eat and after handling raw meat, soil, sand or cats. · Avoid rubbing your eyes or face when preparing food, and wipe the counter clean afterwards. Avoid eating raw eggs and drinking unpasteurized milk.
Giardiasis	metronidazole (Flagyl)/ furazolidone (Furoxone)/ paromycin (Humatim), / quinacrine (Atrabine)	· Frequent washing of the hands, including under the fingernails · Not eating unwashed fruits unless peeled · Boiling water if there is any doubt about its source · Boiling stream or river water for three minutes · Using filter paper with pore size of 1 micro <>
Malaria	Metronidazole/ Nitazoxanide/ Furazolidone/ Tinidazole/ aminoglycoside paromomycin/ chloroquine/ mefloquine(lariam)/ primaquine, quinine/ pyrimethamine-sulfadoxine (Fansidar)/ doxycycline/ artemisin- derivatives/ atovaquone-proguanil (malarone)	· evaluating the risk of exposure to infection · preventing mosquito bites by using DEET mosquito repellant, bed nets, and clothing that covers most of the body chemoprophylaxis (preventive medications)
Leishmaniasis	Na stibogluconate (Na antimony gluconate) / meglumine antimonite/ liposomal amphotericin/ pentamidine/ itraconazole/ Fluconazole/ Paromomycin/ miltefosine (impavido)	insect repellents containing DEET provide protection. Insect screens, bed nets,and clothing are more effective if treated with permethrin or pyrethrum, because the tiny flies can penetrate mechanical barriers. Vaccines are not currently available. Wear long selves shirt and long pants and sock.(tuck in shirt into the pants)

References

http://www.itg.be/itg/DistanceLearning/LectureNotesVandenEndenE/13_Arbovirusesp5.htm#T10
http://www.medicinenet.com/leishmaniasis/page4.htm

MMIC PBL 2

2008-01-21T08:39:00.000-08:00

Virus

Microbe	Transmission	Signs and symptoms	Disease	Geographical distribution
Hepatitis A virus	Fecal-oral route	Diarrhea and nausea with possible acute infection	Hepatitis A	Worldwide, but most common where sanitary conditions are poor and the safety of drinking water is not well controlled
Hepatitis B virus	Parenteral route	Similar to HAV with higher possibilities of leading to chronic infections and liver cancer	Hepatitis B	Worldwide, but with differing levels of endemicity. In north America, Australia ,northern and western Europe and New Zealand, prevalence of chronic HBV infection is relatively low (less than 2% of the general population)
Nairovirus	Bite from Hyalomma tick, animal reservoirs	Fever, shaking chills, severe headache, rash and possible hepatomegaly	Crimean- Congo Hemorrhagic Fever	Nil
Viruses belonging to the Flaviviridae family	Bite from Mosquitoes ( eg. Culex tritaeniorhynchus) or Ticks, Birds serve as the reservoir	Fever, drowsiness, facial flushing, lymph node Largement, conjunctival infection	West Nile Fever	Nil
Flavi- viruses	Bites from mosquitoes, most commonly Aedes aegypti, a day biting mosquito	Dengue hemorrhagic fever, rash, headache, respiratory manifestations that mimic a cold	Dengue	Dengue fever is widespread in tropical and subtropical regions of central and south America and south and south-east Asia and also occurs in Africa; in these regions , dengue is limited to altitudes below 600 metres (2,000 feet)
Alphavirus	Bite from Culex mosquito , Birds serve as the reservoir	Fever, rash and polyarthritis	Sindbis	Nil
Phlebovirus	Bite from mosquitoes, exposure to infected animals and possible aerosol	Nonspecific febrile reaction with fever, nausea and possible visual loss	Rift Valley Fever	Nil
Viruses from Rhabdoviridae family	Open cuts or wound in skin or mucous membranes via bites or infected animal saliva	Fever, severe headache, malaise ,possible neurologic Manifestations	Rabies	Rabies is present in animals in many countries worldwide. Most cases of human infection occur in developing countries
Phlebo- viruses	Bites from sandfly (Phleobotomus papatasi)	Fever, severe frontal headache, nausea, vomiting, possible aseptic Meningitis	Sandfly fever	Nil
Leishmania- viruses	Leishmania species	Infection with Leishmania	Leishmania- virus	Nil
Japanese encephalitis (JE)virus, which is a flavivirus	The Japanese encephalitis virus is transmitted by various mosquitoes of the genus Culex	asymptomatic (e.g. cause no symptoms). In symptomatic cases, severity varies ;mild infections are characterized by febrile headache or aseptic meningitis	Japanese encephalitis	Nil
an arbovirus of the Flavivirus genus	Bites from mosquitoes (Aedes aegypti)	asymptomatic, most lead to an acute illness characterized by two phases. Initially, there is fever, muscular pain, headache, chills, anorexia, nausea and/ or vomiting, often with bradycardia	yellow fever	The yellow fever virus is endemic in some tropical areas of Africa and central and south America. The number of epidemics has increased since the early 1980s. Other countries are considered to be at risk of introduction of yellow fever due to the presence of t he vector and suitable primate hosts (including Asia, where yellow fever has never been reported)

Fungal

Causes of FUNGUS DISEASES
Fungal infection outbreak could happen in a clinical setting where climate are often hot and high humidity such as tropical areas like Indonesia. These diseases are caused by poor personal health practices (harsh living conditions of NS soldier). The jungle environment promotes fungus and bacterial diseases of the skin and warm water immersion skin diseases. Bacteria and fungi are tiny plants which multiply fast under the hot, moist conditions of the jungle. Sweat-soaked skin invites fungus attack. The following are common skin diseases that are caused by long periods of wetness of the skin:

Warm Water Immersion Foot. This disease occurs usually where there are many creeks, streams, and canals to cross, with dry ground in between. The bottoms of the feet become white, wrinkled, and tender. Walking becomes painful.
Chafing. This disease occurs when soldiers must often wade through water up to their waists, and the trousers stay wet for hours. The crotch area becomes red and painful to even the lightest touch.
Most skin diseases are treated by letting the skin dry.

Microbe	Transmission	Signs and symptoms	Disease	Geographical distribution
Dermatophytes, e.g: Microsporum, Trichophyton, epidermophyton sp	Direct contact with skin, usually on warm, sweaty and humid part of the body, hair and nails,feet.	typical ringworm lesion, itching, scaling, inflammation, and blisters	tinea capitis(scalp ringworm), tinea cruris(jock itch) and tinea pedis(athelete’s foot)	contact with infected lesions, soiled or contaminated articles such as shoes and towels, almost anywhere in the world
Sporothrix schenckii	mold spores enter skin in puncture wounds by thorns in jungle	small bumps on skin but painless, local abscess and ulcerative nodules	sporotrichosis	soil, wood, sphagnum moss, and decaying vegetation throughout the world
Histoplasma Capsulatom	Inhalation of airborne asexual pores causing acute respiratory diseases	Often no symptoms but fever, cough, malaise, respiratory symptoms can occur.	Histoplasmosis	grows preferentially in soil enriched with bird droppings
Candida Albicans	Part of the normal flora of skin, mucous membrane and GI tract, can invade tissue if the infected person is injured or wounded	: itchy skin rash, skin inflammation, skin lesions on moisture-damaged skins, rash in e penis area	disseminated candidiasis and chronic mucocutaneous candidiasis	Human body
Aspergillus fumigatos	Inhalation of airborne spores and their invasion through a wound or other tissue injury	Fever, cough, pneumonia, endocarditis, eosinophilia	Aspergillosis	live in soil, commonly in decaying vegetation, such as fermenting compost piles and damp hay.
Cryptococcus neoformans	Inhalation of airborne yeast cells, affects the central nervous system and the lungs in people with weakened immune systems.	early lung infection often has no symptoms, meningitis, encephalitis and headache.	Cryptococcosis, especially Cryptococcal meningitis	live in soil, especially when enriched with pigeon droppings

Prevention of FUNGUS DISEASES in soldiers
To prevent these diseases, soldiers should:

Bathe often, and air- or sun-dry the body as often as possible.
Wear clean, dry, loose-fitting clothing whenever possible.
Not sleep in wet, dirty clothing. Soldiers should carry one dry set of clothes
just for sleeping. Dirty clothing, even if wet, is put on again in the morning.
This practice not only fights fungus, bacterial, and warm water immersion
diseases but also prevents chills and allows soldiers to rest better.
Not wear underwear during wet weather. Underwear dries slower than jungle fatigues, and causes severe chafing
Take off boots and message feet as often as possible.
Dust feet, socks, and boots with foot powder at every chance.
Always carry several pairs of socks and change them frequently.
Keep hair cut short.

Protozoa

Microbe	Transmission	Signs and symptoms	Disease	Geographical distribution
Leishmaniasis species	sand fly	fever, damage to the spleen and liver, and anaemia, Visceral/ Cutaneous/ Diffuse cutaneous /Mucocutaneous leishmaniasis	Leishmaniasis	Jungle setting: large number of mosquitoes and sand fly
Plasmodium falciparum and malariae	bites from Female anopheline mosquito	Moderate to severe shaking chills High fever, Profuse sweating as body temperature falls, General feeling of unease and discomfort, (malaise), Headache ,Nausea, Vomiting, Diarrhea	Malaria	Occur in many tropical and sub-tropical countries- P. falciparum and P. malariae is most common in Asia
Giardia	Contaminated food and water Raw food like fruits and vegetables	Abdominal pain, Watery diarrhea Foul smelling gas and burping, Mild fever, sometimes with chills, Malabsorption, where nutrients are not absorbed, frequently with weight loss	Giardiasis	Found in surface waters all over the Earth and spread in the feces of both humans and animals
Toxoplasma gondii	Contaminated food and water Raw food like fruits and vegetables	headache, chronic malanise, fever lymphadenopathy	Toxoplasmosis	almost everywhere from surface of water to contaminated veggies and fruits to soil and infects warm blooded vertebrates
Cryptosporidium	Contaminated food and water Raw food like fruits and vegetables /faecal-oral route or swimming	Dehydration, Malnutrition, Weight loss, Stomach cramps or pain, Fever, Nausea , Malaise , Vomiting	Cryptospori- diosis	Found in surface waters all over the Earth and spread in the feces of both humans and animals
Cyclospora cayetanensis	Contaminated food and water Raw food like fruits and vegetables	Headache, Nausea, Vomiting, sever Diarrhea, bloating, muscle aching, fatigue and asymptomatic	Cyclosporiasis	mainly in America and Canada
Entamoeba histolytica	Contaminated food and water Raw food like fruits and vegetables	sever diarrhea, abscesses in the intestine, liver, and other organs	Amebiasis	Entamoeba histolytica is endemic in tropical countries ,usually found in water, decaying organic matter, soil, and sewage, is of particular interest to contact lens wearers
Naegleria fowleri	nasal passage via swimming and diving	disease primary amebic meningoen- cephalitis (PAM), a brain inflammation	Naegleria infection	found in environment water and soil worldwide, commonly in Warm bodies of freshwater, such as lakes, rivers, Geothermal water such as hot springs, Warm water discharge from industrial plants, Poorly maintained and minimally chlorinated swimming pools
Balantidium coli	Contact with pig feces or soil contaminated with pig feces	severe diarrhea and intestinal abscesses	Balantidiasis	common in tropical regions and many developing countries in the tropical region lack proper water structures for much of the poor, rural population such as Bolivia, the Philippines, and Papua New Guinea
Vibrio cholerae	Faceal-oral route or contaminated food and drink sand more prevalent in warmer cilmate	sever diarrhoea, dehydration, shock , muscle cramps , nausea and vomitting	Cholera	Cholera is most common in Africa, southern and Southeast Asia, and the Middle East, although outbreaks have occurred in Japan , Australia, and Europe
S.japonicum, S.mekongi, S.mansoni, S.intercalatum and S.haematobium	faecal oral route	Katayama fever, abdominal pain, hematuria, weakness, headaches , joint and muscle pain, diarrhea, nausea, producing seizures or transverse myelitis as a result of mass lesions of the brain or spinal cord and cough	Schistoso- miasis	This infection occurs widely throughout the tropics and subtropics

References

http://www.microbiologybytes.com/iandi/6b.html

http://www.mayaparadise.com/diseasee.htm

http://www.nuim.ie/staff/dpringle/courses/mg/chapter02.pdf

http://human-infections.suite101.com/article.cfm/protozoan_parasites_in_dirt\

http://www.who.int/water_sanitation_health/dwq/admicrob5.pdf

Medical Microbiology- PBL 1 ( second blog)

2007-12-09T08:52:00.000-08:00

Case study 1
List of microbes: Staphylococcus aureus, Staphylococcus saprophyticus, Enterococcus faecalis. Escherichia coli, Enterobacter, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Chlamydia trachomatis.

A gram stain will be done first for the suspected bacteria that can cause UTI, before proceeding to any laboratory investigation.

Gram positive bacteria: Staphylococcus aureus, Staphylococcus saprophyticus, Enterococcus faecalis.Gram negative bacteria: Escherichia coli, Enterobacter, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Chlamydia trachomatis.

Biochemical and Culture Testing

Possible Organisms	Staphylococcus aureus	Staphylococcus saprophyticus	Enterococcus faecalis
Gram Stain :positive	cocci(clusters)	cocci(clusters)	cocci(chains)
Culture on Sheep blood agar	haemolytic, yellow colonies	non-haemolytic white colonies	gamma non-haemolytic white colonies( but can show weak alpha haemolysis)
Catalase test	positive	positive	negative
Coagulase test	positive	negative	nil

Possible Organisms	Escherichia coli	Enterobacter sp	Pseudomonas aeruginosa	Klebsiella pneumoniae	Proteus mirabilis
Gram Stain: negative	bacilli	bacilli	bacilli	bacilli	bacilli
Culture on Mac Conkey	pink lactose fermenting colonies	very weak lactose fermenters	non-lactose fermenting colonies producing blue-green pigments	pink lactose fermenting colonies	non-lactose fermenting colonies
Culture on eosin methylene blue (EMB)	metallic green sheen with dark colonies	brown-centered with pale blue colonies	colorless colonies indicating no lactose fermentation and acid production	brown dark-centered colonies indicating lactose fermentation and acid production	colorless colonies indicating no lactose fermentation and acid production
oxidase	negative	negative	positive	negative	negative
*Triple sugar iron (TSI)	acidic slant/acidic deep	alkaline slant/acidic deep	No change	alkaline slant/acidic deep	alkaline slant with black precipitate

acidic slant/acidic deep: ferment lactose and glucose
alkaline slant/acidic deep : ferment glucose only
No change: no carbohydrate fermentation
Black precipitate: H2S production

If oxidase test is negative, proceed to IMViC biochemical test

IMViC	Escherichia coli	Enterobacter sp	Klebsiella pneumoniae	Proteus mirabilis
Indole	+	-	-	-
Methyl red	+	-	-	+
Voges proskauer	-	+	+	-
Citrate test	-	+	+	+
Urease	-	-	-	+

Antibiotic Susceptibility test
5 antibiotics: Gentamycin, Ceftadizime,Cefuroxime, Ampicillin and Ciprofloxacin. Varying zone diameter size can be observed for both the gram positive and negative bacteria.

If Chlamydia trachomatis is highly suspected, as it is a common STD that can cause UTI, some portion of the urine sample can be send for DNA based analysis method such as polymerase chain reaction(PCR).

Case study 2 Besides Salmonella, there are also several other possible microorganisms that could lead to enterocolitis or cause the diarrhea in the patient. Here are the other possibilities:
1. Enterotoxigenic Escherichia coli
2. Campylobacter jejuni
3. Clostridium difficileShigella: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei

Type of microbe	Microscopy test	Biochemical Test	Serology test	Culture
Salmonella	Gram Stain: Gram negative bacilli	TSI: alkaline slant/acid butt with H2S production Indole: Negative Methyl-red: Positive Voges-Proskauer: Negative Citrate: Positive	Slide agglutination test: serotyping using O, H and Vi antigens Tube agglutination test: detect agglutinating Ab to O & H Ag in patient’s serum	MacConkey agar: Observe plate for non-lactose fermenting (clear) colonies Hektoen agar: Observe plate for clear or green colonies and colonies with black centers (H2S production) Salmonella-Shigella agar: Observe plate for clear colonies and colonies with black centers (H2S production) XLD Agar: Observe plate for red colonies and colonies with black centers (H2S production)
Shigella	Gram Stain: Gram negative bacilli	TSI: Alkaline slant/acid butt but no H2S production Indole: Negative Methyl-red: Positive Voges-Proskauer: Negative Citrate: Negative	Slide agglutination test	MacConkey agar: Observe plate for non-lactose fermenting (clear) colonies Salmonella-Shigella agar: Observe plate for clear colonies and colonies WITHOUT black centers (no H2S production) Hektoen agar: Observe plate for clear or green colonies and colonies WITHOUT black centers (no H2S production)
Enterotoxigenic E.Coli	Gram Stain: Gram negative bacilli	TSI: Alkaline slant/acid butt with gas but not H2S production Indole: Positive Methyl-red: Positive Voges-Proskauer: Negative Citrate: Negative	Serotyping using O & H Ag	MacConkey agar: Observe plate for red/pink colonies (lactose-fermenting colonies) EMB agar: Observe plate for greenish metallic sheen
Campylobacter jejuni	Gram Stain: Gram negative bacilli that appear either comma or S- shaped	TSI: Alkaline slant/deep Oxidase: Positive	nil	Selective “CAMP” agar at 42ºC in microaerophilic environment (grow at 5% oxygen + 10% carbon dioxide)
Clostridium difficile	Gram Stain: Gram positive bacilli	nil	nil	Blood agar at human body temperatures

Antibiotic susceptibility testing:
1. Enterotoxigenic Escherichia coli:
• Ampicillin
2. Campylobacter jejuni
• Erythromycin
3. Shigella:
• Ampicillin
4. SalmonellaAmpicillin

Case study 3

Laboratory investigations:
Urine culture is to test to identify the exact type of bacteria causing infection.
Culture on:
1.Blood Agar Plate (BAP)
2.Eosin Methylene Blue (EMB) agar
3.MacConkey Agar (MAC)
4.Ordinary nutrient agar
5.Triple Sugar Iron (TSI) agar
All are grown under anaerobic conditions except for Pseudomonas spp. such as P. aeruginosa as it is a strict aerobe.

Microscopy
1.Gram stain
2.Fungal stain
Morphology are studied in terms of the microorganisms’ shape, arrangement, response to strain and specific structures.
Biochemical tests are done to indicate the presence or absence of enzyme(s), a group of enzymes or a whole metabolic pathway. This helps to identify microorganisms.

	Gram staining	Cultures (Under anaerobic conditions)	Biochemical tests	Antibiotic Susceptibility test
Escherichia coli	Gram-negative (pink) bacillus	1. Blood agar: Gamma hemolysis 2. Eosin Methylene Blue agar: Colonies with metallic green sheen 3. MacConkey agar: Pink colonies	1. Indole test: Positive 2. Methyl Red (MR) test: Positive 3. Voges-Proskauer (VP) test: Negative 4. Simmon’s citrate test: Negative 5. Oxidase test: Negative 6. Urease: Negative 7. TSI acid slant/acid butt with gas, no H2S	-Susceptibility depends on the type of strains - Beta-lactamase resistant strains are not sensitive to penicillin and cephalosporin - Non-resistant strains are sensitive to ampicillin and trimethoprim-sulfamethoxazole
Enterococcus faecalis	Gram-positive (purple) cocci	1. Blood agar: Non hemolytic 2. MacConkey agar: Pink colonies with mucoid appearance 3. Bile Esculin Agar: Ferric citrate indicator will turn black	1.Indole test: Negative 2. Voges-Prokauer test: Positive	-Resistant to aminoglycoside, penicillin and vancomycin when given individually -A synergistic combination of aminoglycoside and cell wall-active antibiotics such as ampicillin and vancomycin
Klebsiella pneumoniae	Gram negative (pink) bacillus, a large capsule can be observed	1. MacConkey agar: Pink colonies with mucoid appearance	1. Indole test: Negative 2. Methyl Red test: Negative 3. Voges-Prokauer test: Positive 4. Urease test: Positive	- Isolates from nosocomial infections are frequently resistant to multiple antibiotics - Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)
Pseudomonas aeruginosa	Gram-negative (pink) bacillus	1. Blood agar: Beta-hemolysis 2. MacConkey agar: Colourless colonies 3. Ordinary nutrient agar: Blue-green colonies	1. Indole test: Negative 2. Methyl Red test: Negative 3.Voges -Prokauer test: Negative 4. Catalase test: Positive 5. Oxidase test: Positive 6. TSI agar: Negative (Growth with typical metallic sheen) 7. Pyocyanin test: Positive 8. Urease test: Positive/Negative 9. Fluprescein test: Positive	- Highly multidrug resistant - Combination therapy: Penicillin derivatives, Ceftazidime, Ciprofloxacin, Aztreonam, Imipenam
Serratia marcescens	Gram-negative (pink) bacillus	1. MacConkey agar: Pink colonies 2. Ordinary nutrient agar: Red colonies	1. Indole test: Negative 2. Methyl Red test: Negative 3. Voges-Prokauer test: Positive 4. Urease test: Negative	- Antibiotic resistance vary greatly - Isolates from nosocomial infections are frequently resistant to multiple antibiotics - Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)
Proteus mirabilis	Gram-negative (pink) bacillus	1. Blood agar with phenylethyl alcohol: Colonies do not have swarming effect 2. MacConkey agar: Colourless colonies	1. Indole test: Negative 2. Methyl Red test: Positive 3.Voges-Prokauer test: Negative 4. Catalase test: Positive 5. Urease test: Positive 6. TSI agar: Black butt	- Sensitive to ampicillin, aminoglycosides and trimethoprim sulfamethoxazole

Case Study 4
1.Chlamydia pneumoniae
• Obligate intracellular bacterium
• Does not gram stain
• Affects adults and children

2. Haemophilus influenza
• Pleomorphic gram-negative bacillus
• Affects children and adults (especially with COPD-Chronic Obstructive Pulmonary Diseases)

3. Moraxella catarrhalis
• Oxidase positive
• Gram-negative diplococcus
• Affects children and adults with COPD

4. Pseudomonas aeruginosa
• Glucose-nonfermenting
• Gram-negative bacillus
• Affects adults and children, diabetic adults, nosocomial, CF (Cystic Fibrosis) patients

5. Streptococcus pneumoniae
• Gram-positive lancet-shaped cocci
• Appear in pairs or short chains
• Affects adults (mainly elderly)

6. Mycoplasma pneumoniae
• Smallest free-living organism
• Lacks a bacterial cell wall
• Does not gram stain

7. Staphylococcus aureus
• Gram-positive cocci in clusters
• Coagulase-positive
• Catalase-positive
• Produces Beta-lactamase

8. Paragonimus westermani
• Fluke (Trematode)
• Affects children and adults in endemic areas

9. Adenovirus
• Enveloped dsDNA (double-stranded DNA)
• Affects children and adults

10. Parainfluenza virus Type I, II, III
• Enveloped ssRNA (single-stranded RNA)
• Affects infants and young children

11. Bordetella pertussis
• Coccobacillary, encapsulated gram-negative rod
• Negative blood culture

Lab investigations

Wet mounts
• Observe for microbe structure – bacillus, cocci, lancet-shaped, size, etc.

Gram stain
• Positive gram stain – microbe will stain purple/ blue
• Negative gram stain – microbe will stain red/ pink

Acid-fast bacterium stain
• Stains mycobacterium that do not gram-stain due to their high lipid content

Direct fluorescent-antibody stain
• Histologic stain to detect spirochetes

Peripheral blood films
• Observe microbial activity in blood
• Most respiratory tract infections would have negative blood smears

Enzyme immunoassay
• Identifies organisms with known antiserum
• Specific antibody linked to its homologous antigen

Latex agglutination assay
• Latex beads coated with specific antibody
• Agglutination will occur in the presence of the homologous bacteria

Blood cultures
• Positive blood culture – microbial growth (gold, yellow colonies, etc.)
• Negative blood culture – no microbial growth

Bacteriologic sputum cultured on enriched agar
• Bordet-Gengou agar

Antibiotic susceptibility tests

Penicillin – a general antibiotic for penicillin sensitive isolates
Ceftriazone
Erythromycin
Tetracycline – eg. Doxycycline
Praziquantel

Case study 5
Possible microorganisms

Microorganism Test Result
Staphylococcus aureus
● Gram-staining

● Culturing on mannitol salt/blood

agar

● Coagulase test

● Catalase test

● TSI

● Gram-positive, cluster-forming cocci

● Yellow or gold

colonies, drop in pH (yellow area) / ß-hemolytic

● Positive

●

Positive

● Acid slant/acid butt
Enterococci

● Gram-staining

● Catalase test

● Gram-positive cocci, occuring singly, in pairs, or in short

chains
● Negatives
Coagulase-negative staphylococci
● Gram-staining

● Culturing on blood agar

●

Coagulase test

● Catalase test

● Gram-positive, cluster-forming coccus

● Yellow or

gold colonies

● Negative

● Positive
Escherichia coli
● Gram-staining

● Culture on EMB/ MacConkey's agar

●

TSI agar

● Urease test

● Indole test

● Citrate

test

● Gram-negative rod

● EMB:Lactose-fermenting, blue-black

colonies with metallic green sheen

● MacConKey:Lactose-fermenting, red

colonies

● Acid slant/acid butt with gas but no H2S

● Negative●

Positive

● Negative
Pseudomonas aeruginosa
● Gram-staining● Culture on EMB/ MacConkey's agar

● TSI●

Oxidase test

● Indole test

● Citrate

● Gram-negative rod

● EMB/MacConkey:Non-lactose fermenting

colonies

● Alkaline slant/alkaline butt

● Positive

●

Negative

● Positive
Enterobacter species
● Gram-staining

● Culture on EMB/ MacConkey's agar

●

Urease test

● Vogues-Proskauer test

● Citrate test

● Gram-negative rod

● EMB: Lactose-fermenting, brown dark

-centered, mucoid colonies

● MacConkey:Lactose-fermenting, pink mucoid

colonies

● Negative

● Positive

● Positive
Proteus mirabilis
● Gram-staining

● Culture on EMB/ MacConkey's agar

●

TSI

● IMVIC

● Urease test

● Gram-negative cocci

● EMB/MacConkey:Non-lactose fermenting

colonies

● Alkaline slant/acid butt with H2S

● Indole:

Negative

● Methyl-red: Positive

● Vogues-Proskauer: Negative

● Catalase: Positive

● Positive
Klebsiella pneumoniae
● Gram-staining

● Culture on EMB/ MacConkey's agar

●

TSI

● Indole test

● Urease test

● Citrate test

● Gram-negative rod

● EMB: Lactose-fermenting, brown dark

-centered, mucoid colonies

● MacConkey:Lactose-fermenting, pink mucoid

colonies

● Acid slant/acid butt with some gas production, no H2S

●

Negative

● Positive

● Positive

Antibiotic:
Methicillin
Vancomycin
Penicillin
Oxacillin

Case study 6
Antibiotic Susceptibility test 5 antibiotics: vancomycin, ciprofloxacin , Erythromycin, bactrim and cefamandole

Gram-negative Test Result
Gardnerella vaginalis
● Morphology

● Oxidase

● TSI

● IMViC

● Laboratory diagnosis /culture

●Bacilli

● negative

● Acidic slant/acidic deep

● Catalase (-)

● Chocolate agar and HBT agar: Small, circular, convex, gray colonies

●Colistin-oxolinic acid blood agar: Beta-hemolysis
Escherichia coli
● Morphology

● Oxidase

● TSI

● IMViC

● Laboratory diagnosis /culture

●Bacilli

● negative

● Acidic slant/acidic deep

● I(+),M(+), Vi(-),C(-), U(-)

● EMB: green sheen,fermenting colonies

●MacConkey agar: fermenting colonies
Neisseria gonorrhoeae
● Morphology

● Oxidase

● TSI

● Laboratory diagnosis /culture

●Bacilli

● positive

● Acidic slant/acidic deep

●Giemsa-stained

●PCR and ELISA

●Immunofluorescence
Chlamydia trachomatis
● Morphology

● Oxidase

● Laboratory diagnosis /culture

●cocci

● positive

●Giemsa-stained

●PCR and
ELISA

●Immunofluorescence
Pseudomonas aeruginosa
● Morphology

● Oxidase

● TSI

●
IMViC

● Laboratory diagnosis /culture

●Bacilli

● positive

● Alkaline slant/ alkaline butt

●Catalase (+)

●EMB and MacConkey agar: non-fermenting colonies

Gram - positive Enterococcus faecalis Staphylococcus saprophyticus
Morphology Cocci( in pairs) Bacilli
Catalase test - +
coagulase Nil -
Laboratory diagnosis /culture Blood agar: non-hemolysis Mac Conkey’s agar: Spherical, irregular grape-like cluster in culture

Other microbes Trichomonas vaginalis Candida albicans Mycoplasma hominis
Morphology acridine orange : pear-shaped, motile, flagellated protozoan single-celled, diploid fungus round, pear shaped and even filamentous
Laboratory diagnosis /culture
Trichomonas Direct Enzyme Immunoassay and Fluorescent Direct Immunoassaysaline

wet preparation : motile trichomonads and increased PMNs (ratio of PMNs to vaginal epithelial cells)

Blood agar plates: large, round, white or cream colonies Mycoplasma GU Culture System: ‘fried egg’ and granular appearance colonies

Medical Microbiology- PBL 1

2007-12-02T05:20:00.000-08:00

Learning issues:
1. Define the possible diagnosis
2. List down the possible causative agent

Case Study 1
Patient: Female/27 years old
Signs and symptoms: Fever, chills and dysuria
Suspected Diagnosis: Urinary tract infection
Specimen collected: Urine

The female patient in case 1 was diagnosed with urinary tract infection. UTI occurs due to the infection of microorganisms in the bladder,urethra and kidneys. It can be further classified as cystitis( bladder infection) or pyelonephritis( kidney infection). The patient showed common UTI symptoms seen in cystitis like dysuria(painful urination), fever and chills, but did not complain of back pain, or haematuria, hence it is likely that she is suffering from cystitis.

UTI occurs more frequently in woman as compared to men, due to the short urethra they have, allowing entry of bacteria into the urinary tract. The most common cause of UTI is Escherichia coli, a normal flora in the intestine and colon that enters and invades the urethra causing an infection. The second most likely bacteria that can cause UTI is Staphylococcus saprophyticus, as it usually infects woman in between the age of 20-40. Other microbes such as Klebsiella pneumoniae, Proteus mirabilis and Enterococcus species can also cause UTI, however, their occurrence is very low .

They are eliminated because:
a) Enterocoocus species usually occurs in patients who have undergone urinary tract surgery.
b) Proteus mirabilis often infect recurrent UTI patients that have structural abnormalities in their urinary system.
c) Klebsiella pneumoniae is involved in hospital-acquired infection and commonly infect immunocomprised individuals.

Most possible diagnosis: Escherichia coli and Staphylococcus saprophyticus
All the above characteristics were not observed in the patient and hence can be eliminated.
The two most likely cause of UTI are Escherichia coli and Staphylococcus saprophyticus.

Case Study 2
Patient: Female/29 years old
Signs and symptoms: Diarrhea
Suspected Diagnosis: Enterocolitis
Specimen collected: Stool

Types of enterocolitis:
1) Necrotizing enterocolitis
●Gastrointestinal disease that mostly affects premature infants, NEC involves infection and inflammation that causes destruction of the bowel intestine or part of the bowel.
● NEC typically occurs within the first 2 weeks of life, usually after milk feeding has begun

2) Autistic enterocolitis
●Autistic enterocolitis is a controversial term first used by British gastroenterologist Andrew Wakefield to describe a number of common clinical symptoms and signs which he contends are distinctive to autism.
● The existence of autistic enterocolitis is controversial, as the methodology of Wakefield's studies has been criticized and his results have not been replicated by other groups

3) Salmonella enterocolitis
● Most common type of food poisoning
● Infection in the lining of the small intestine caused by the bacteria Salmonella.
●Symptoms include diarrhea and abdominal pain

Most possible diagnosis: Salmonella enterocolitis
Reasons: Since necrotizing enterocolitis affects mainly infants but the patient is a 29-year-old female, it is highly unlikely that she is suffering from this disease. Moreover, it is not known to the medical technologist that the patient is suffering from autism. Hence, there is very low chance of her suffering from autism enterocolitis. The symptoms of salmonella enterocolitis include diarrhea which is one of the complaints as told by the female patient.

Case Study 3
Patient: Female/67 years old
Signs and symptoms: Fever, chills, bladder distension (bladder stretching); on indwelling catheter
Suspected Diagnosis: Urinary tract infection
Specimen collected: Urine

Indwelling catheters
Indwelling catheters avoid distension by emptying the bladder continuously into a bedside drainage collector. Individuals with indwelling catheters are encouraged to maintain a high fluid intake in order to prevent bacteria from accumulating and growing in the urine.

Possible agents:
● Many different Gram-negative organisms colonize urinary catheters, often becoming invasive infections.
●The most commonly isolated pathogens are Escherichia coli and Enterococcus spp.E.coli uses fimbriae to adhere to the urinary epithelium, thereby reducing the risk of being washed away.
● Infections caused by Proteus spp. are more likely in patients who have stones as Proteus spp. have urease activity that raises urinary pH, thus encouraging stone formation.
● Staphylococcus saprophyticus is a common isolate from sexually active females.
● Other intestinal bacteria, including Klebsiella pneumoniae (K.pneumoniae), Proteus mirabilis (P.mirabilis) , and Citrobacter.
●Others include Pseudomonas aeruginosa (P.aeruginosa), Enterobacter, and Serratia species, gram-positive organisms, including Enterococcus species, and S. saprophyticus .

Most possible diagnosis: Staphylococcus saprophyticus, Klebsiella pneumoniae, Proteus mirabilis and Enterococcus
UTI in this patient should be due to the presence of the catheter in the urethra. Hence these microbes might the possible reason.

Case Study 4
Patient: Male /68 years old
Signs and symptoms: fever, chills, excessive phlegm, breathing problems Suspected Diagnosis: Bronchitis
Specimen collected: Sputum

Information on Bronchitis
Inflammation of the mucous membrane in the lungs' bronchial passages
Narrowed bronchial passages shuts off the tiny airways in the lungs
Results in coughing spells, thick phlegm and breathlessness
Two forms: acute (lasts less than 6 weeks) and chronic (more than two years)

Acute bronchitis
●responsible for the hacking cough and phlegm production that sometimes accompany an upper respiratory infection
● In most cases the infection is viral in origin, but sometimes it's caused by bacteria
● very common among both children and adults

Chronic bronchitis
● a serious long-term disorder that often requires regular medical treatment

Possible agents
1) Adenovirus
● Non-enveloped double-stranded linear DNA
● Icosahedral nucleocapsid with a fiber protruding from each of the 12 vertices
●Causes bronchitis when it affects the lower respiratory tract

2) Bordetella
●Small, coccobacillary, encapsulated gram negative rod
● Restricted to the respiratory tract (negative blood culture)
●Isolated and grown on Border-Gengou agar

3)Parainfluenza virus
●Single stranded RNA negative-strand viruses

4) Streptococcus pneumoniae
● Gram positive lancet-shaped cocci
● Arranged in pairs or short chains
●Higher mortality in persons aged 65 and above

5) Chlamydia pneumoniae
●Obligate intracellular bacteria
●Require host cells for growth
● Causes upper and lower respiratory tract infections

Most possible microbes:
These microbes expressed similar symptoms as the patient in this case study. Hence, they are the most likely microbes to be causing this illness.

Case Study 5
Patient: Male /37 years old
Signs and symptoms: fever, Swelling around operation wound
Suspected Diagnosis: Wound Infection
Specimen collected: wound swab

Wound Infection caused by bacteria
● This is because swelling is one of the hallmarks of inflammation due to infection by either endogenous factors like tissue necrosis or exogenous factors like microorganism infections.
● Fever, on the other hand, is a common manifestation of infection and inflammation that is caused by many bacterial products eg endogenous or exogenous pyrogens.
● Most wound infections are caused by normal flora found on the skin/body.

Pathogens related to different surgical procedures/operations:

Pathogens Commonly Associated with Wound Infections and Frequency of Occurrence are as follows:
● Staphylococcus aureus (20%)
● Coagulase-negative staphylococci (14%)
● Enterococci (12%)
● Escherichia coli (8%)
● Pseudomonas aeruginosa (8%)
● Enterobacter species (7%)
● Proteus mirabilis (3%)
● Klebsiella pneumoniae (3%)
● Other streptococci (3%)
● Candida albicans (3%)
● Group D streptococci (2%)
● Other gram-positive aerobes (2%)Bacteroides fragilis (2%)

Most possible microbes:
However, out of all the organisms, the most common bacteria involved in wound infection due to operation was found to be Staphylococcus aureus, which accounts for 17-20% of the cases reported. Out of these cases, 40-50% are due to MRSA (Methicillin-resistant Staphylococcus aureus).

Case Study 6
Patient: Female/37 years old
Signs and symptoms: Fever, pain during urination and virginal discharge
Suspected Diagnosis: Urinary tract infection
Specimen collected: Vaginal discharge

Possible agents:
1) Trichomoniasis
A sexually transmitted disease caused by the anaerobic, flagellated protozoa Trichomonas vaginalis.
Symptoms of Trichomoniasis include painful urination, fever, discharge greenish-yellow vaginal fluid, lower abdominal pain and discomfort during sexual intercourse.

2) Bacterial Vaginosis
It is caused by imbalance of bacteria flora in vagina. Usually, it is characteristic by the overgrowth of Gardnerella vaginalis and Gardneralla mobiluncus,.
Gardnerella bacteria is facultative anaerobic and gram-negative, while Mycoplasma hominis
It symptoms includes gray vaginal discharge and painful urination.

3) Vaginal Candidiasis
An infection caused by yeast, Candida albicans.
Its morphology appearance is normally single-celled.
Symptoms includes discomfort during urination and produce cottage cheese-like vaginal discharge or irritation in genital area

4)Gonorrhea
Another sexually transmitted disease caused by Neisseria gonorrhoeae, which is a gram-negative, cocci and aerobic bacteria.
Which produces symptoms like fever, yellowish discharge and urethritis.

5)Chlamydia
A sexually transmitted disease caused by gram-negative cocci and aerobic bacteria Chlamydia trachomatis.
Chlamydia trachomatis required a host organism to survive.
Patients with Chlamydia will experience symptoms like fever, abnormal discharge and painful urination.

Most possible microbes:
Since Gonorrhea, Trichomoniasis and Chlamydia expressed similar symptoms as the patient in this case study, they are the most likely microbes to be causing this illness.

References
1. http://www.mayoclinic.com
2. http://www.healthatoz.com
3. http://www.nlm.nih.gov/medlineplus/encyclopedia.html
4. http://en.wikipedia.org/wiki/Wiki

Week 19 - Quality Assurance & Quality Control

2007-11-06T23:36:00.000-08:00

hey people,im really sorry for the super late posting..for this posting i will share with u guys the quality assurance and quality control in the histopathological laboratory where im attached to.

Quality Assurance(QA) and Quality Control(QC)

QA is a program in which overall activities conducted by the institution are directed towards assuring the quality of the products and services provided. QC is under QA in which products and services provided are evaluated in hopes of achieving high accuracy and reliability of test results and also aids troubleshooting. This involves continuous monitoring of operations and systemic day-today checking of the produced data to decide whether these are reliable enough to be released.

Application of QC protocol to:

Tissue processing
The ATPs are checked against maintenance checklist to ensure that all reagents are properly loaded and in sufficient amounts prior to start of processing. Reagents are maintained (after 20 cycles) to ensure a certain standard of quality of processing in subsequent cases.

Embedding
Paraffin dispensers are emptied monthly to clear debris collected at the bottom of the container.
Microtomy
The temperature of floatation baths are checked and recorded daily on the temperature log as part of equipment maintenance to ensure that it is in top condition during production of results. If out of range (less than 44oC or more than 50oC), corrective actions are taken by adjusting control knob. The microtomes and floatation baths are cleaned daily to remove debris after all sectioning done.

Staining
Working thermometer and equipment temperature display are calibrated monthly to make sure that readings displayed on ovens and refrigerators are within control range. The coldroom used to store special staining kits is inspected daily to ensure that it can function properly. The accuracy of its temperature is checked daily as the activity of special stains will be very much affected by temperature. When preparing staining reagents, the electronic balance may be used to measure out certain ingredients. Therefore, it is calibrated yearly to make sure the reading is accurate and within 1% of the reference mass as certain special stains are sensitive to quantity. Control samples are stained simultaneously with patients’ samples and observed for its quality. This is to ensure that any poor staining caused by defective reagents detected by control samples can be corrected at first hand before the sections are stained. H&E and special stains controls are evaluated and graded according to their quality from 5 (excellent) to 2 and below (inadequate in which remedial actions are to be taken). For H&E control, the nuclear and cytoplasmic control must be well defined and contrasted. As for special stains controls, there must be relatively good demonstration of 75% or more of the cells without background staining. During staining of patients’ samples, 10% of the slides contained in a rack are sent for microscopic examination for grading as part of QA. The slides are evaluated to detect any faults in staining, sectioning and mounting. Percentage of unsatisfactory slides caused by errors in staining, sectioning or mounting are counted monthly as well. Weekly, at the end of every Tuesday and Friday, reagents used for routine H&E are changed as part of QC so that the quality of staining can be consistent throughout. Records are maintained which indicate the type of reagent that is changed, filtered or replaced. Since Haematoxylin and eosin are prepared once a week, they are filtered daily to remove debris left by the previous slides. Before sending the slides for mounting, the machine is primed to remove any air bubbles present as it can affect the microscopic examination and thus, the quality of staining.

Dispatch slides
All slides are checked against tissue blocks prior to their dispatching. If unsatisfactory, they are
sent for recuts. Also, the number of slides indicated on request forms must tally against the amount being stained.

Lastly, pathologists evaluate the results in areas of sectioning, staining and mounting as these are the criteria that will affect the quality of section and thus, hinder diagnosis. The total number of errors that are being raised in all stages of workflow (from processing to labeling) are counted monthly and systematically documented in an ‘EVENTS’ file. In addition, a maintenance program for all equipment and instruments, listing the model and serial number, dates of services performed, parts installed, and the interval and date of the next service are kept and recorded. There is also a complete equipment inventory, listing the name and manufacturer of the instrument, model and serial number, purchased from, personnel who installed the instrument, the date, length of warranty, purchase price , and location of the manual of instructions covering the equipment.

This shall be the end of my posting. thanks for reading. :)

Sharon Ang
0503219H

Week 18 Attachment Sharing

2007-10-28T06:28:00.000-07:00

Alright, this week i'll be sharing more on the analysis, excision and identification of proteins. Also, i'll touch a bit on the DRP group that me and Jiaxin were put in charge of. This weeks blog is more of a reflective entry rather than a factual one.

After 16 weeks of SIP/MP, our group, comprising of me, Jiaxin, Ming Boon and Shahirah, still have not gotten any concrete results for our tabulation. Furthermore, most of our raw data that have been tabulated requires these final pieces of concrete results as evidence that the entire 20 weeks that were spent and the two projects were a success and also provide credibility that the protocols involved in the projects are reproducible. With 4 more weeks left, we crammed all our analysis, protein spot excision and identification of the possible proteins that have been selected.

Protein spot analysis
After extracting our proteins for both the cell membrane and secretome project, we ran a 2-D gel before scanning the gels to acquire the image of the protein spots. The gels are placed in an imager called the Pharos FX Plus which scans the gels that have been stained with SYPRO Ruby or CyDye. The software, Quantity One, was selected to acquire the image on the computer. After getting pictures of the gels on the computer, another software called the PDQuest is used to customize the picture to our wishes. With this software, we are also able to magnify the protein spots that we choose to view such that a 3-D image of the protein spot will be shown on the computer.

For our cell membrane protein project, gels that were run according to experiment numbers would be compared. For instance, Experiments 6, 7 and 8 were protein samples from a selected S. Maltophilia strain and Experiments 9, 10 and 11 were from a different strain. The gels containing protein samples from Exp 6, 7 and 8 would be compared while Exp 9, 10 and 11 would be compared. From there, the best gel in terms of resolution and spot quality would be chosen for spot excision. For the secretome project, a comparison would be done for gels which run the protein samples belonging to bacterium grown at the same temperature.

Protein spot excision
The protein spots would be selected through the PDQuest software. The Shimadzu Xcise would be used for the excision of spots and mounting of protein samples on the MALDI plate for identification. Although the Xcise has many advantages, the main one to be it is the automated spot excision and processing machine, its major disadvantage is that it still is controlled by the operator. Afetr using the Xcise for the second time, i realized that although it is not as tedious as manual spot cutting, processing and mounting, the automated version is still quite tedious. For instance, the spots that have been selected in the PDQuest software has to be re-selected on the computer sonnected to the Xcise machine. Manual calibration has to be done before selecting the spots and following the selection, the programming of the Xcise machine has to be done. Normally, after programming most machines, the operator would be free to perform other tasks. However, with the Xcise, we still have to monitor the automated process as there have been experiences whereby the excised protein spots were not cut properly or the excised protein spots were emptied into the wrong well. Thus, monitoring of the process is still required.

After the excision, processing and mounting, the protein samples would be mounted on the MALDI plate. The MALDI plate is then analyzed by a MALDI TOF/TOF which allows the sequencing of the peptides in the samples that have been spotted on the MALDI plate. From there, all the data has to be carefully tabulated.

After sharing with everyone the work process, I am sure that people will realize the research is never monotonous work and it requires a whole lot of effort from the individual. Also, a lot of thought has to be put in to actually organize all the data that has been established in such a way that would make sense to the readers.

DRP students
For our SIP assignment, we were also suppose to supervise a group of Differential Research Programme students that wereput under our care. Since i have actually performed the experiments that they were suppose to be performing to extract their own proteins, i thought the students would have actually been easy to handle. I was proven wrong.

In the first 2 weeks of them joining the programme, Jiaxin was left to handle the students since i was carrying out some experiments of our own. On the very first day i actually supervised them, the students were not even sure of the steps involved in their protocol. After a full day in the lab, they realized that they had actually performed the extraction wrongly. A few days later, they ran a 1D gel without denaturing their protein samples. Also, i realized that there was very little communication within the group to the extent that the individual performing the intial inoculation does not know the amount of cells that the individual performing the second inoculation has aliquoted. Also, although they kept their log book updated, they can not recall what they did only a few hours before. This could mean that they were actually following the protocol blindly. Thus, i conclude that if anyone so does wish to go into research, the individual should be completely interested in what he or she is doing.

Thus, i have come to the end of my blog. Although this is not a factual entry, i am still open to any questions. And thank you for listening to my complaints. HAHAHA.... See you guys in school soon.

Johanna
0503309G
TG02

Week 17 Attachment Sharing

2007-10-20T23:33:00.000-07:00

heya.......first and foremost i would like to wish all muslim frenz SELAMAT HARI RAYA.......dun forget to collect as many green packets as possible..it might be our last chance...hehehe....aniwei this week i would post something that was shown to me on the 1st day of work......ISOLATION OF PLASMA AND WBC..yah i noe..a bit outdated....

Now2 dun get confuse...yes i am under research but my lab is also involve in clinical stuff....soo yeah it's a mixture of both...so don't get a shock if u hear people in my lab go...quick sequence this first....the person is waiting for drugs to be administered......so back to the story.....

This whole thing is called blood processing and it is done by the research officer...we attachment students are not allowed to do this because our subsequent experiments will depend on this and if we screw up the blood processing....we are soo dead....we can't possibly ask the kind subject who 'donated' his/her blood to us to 'donate' again can we......

Phlebotomist will help us to draw blood from the volunteers (healthy individuals/patients that agreed to take part in our research). Then our counterpart in the clinical trials dept will then deliver the blood to us (the blood is collected in 3 EDTA tube-purple cap). Depending on what study the blood is for....the apropriate components will be isolated.....it is usually plasma, WBC and DNA..unfortunately i'm unable to post up on DNA isolation because we are not shown on tt....perhaps this wk they'll show us........well they have been promising to show us but the time has not come yet......now..after the delivery of blood......the show begins....

A) Isolation of plasma
1. Centrifuge the 3 tubes of blood at 2000 rpm for 10 min
-this separates the plasma from the other blood components
2. Pipette the supernatant (plasma) into the 1.5ml eppendorf tubes.
-pipette as much supernatant as possible but do not disturb the pellet
3. Store the eppendorf tubes at -80oC fridge
**the plasma is used for HPLC experiment to determine the drug level before, during and after infusion to study the drug pharmacodynamics and kinetics. thus plasma is usually collected for studies that involve drugs (usually in diseased subjects/patients to see their response)

B) Isolation of WBC
1.Transfer 3ml of the pellet (from isolation of plasma) into 15 ml tubes
-the pellet is actually RBC
2. Add 9ml of RBC lysis solution to the tubes
-this forms 1:3 blood to lysis solution ratio
3. Incubate the tubes at room temperature for 10 min
4. Centrifuge the tubes at 2000 rpm at 25oC for 10 min
5. Pour away the supernatant
6. Add 1ml of RBC lysis solution and pipette up and down
7. Transfer everything from the tubes to eppendorf tubes
8.Centrifuge the tubes at 3000rpm for 3min at 4oC
9.Centrifuge at 15000rpm for 5 min at 4oC
Discard the supernatant
11. Store the pellet (WBC) at -80oC fridge
**this WBC will be used to isolate the DNA...soo die2 WBC will be collected for any studies since our lab is a very DNA lab....

tt's all for now......unfortunately this is my last post...left 3 more wks of SIP......enjoy it okies and let's fight till the end..............wishing everybody...ALL THE BEST FOR OUR SIP/MP......:D

nur zahirah tg02

Week 16th-SPSS!!

2007-10-17T16:38:00.000-07:00

Gomennasai, for the late posting! Here's my posting for week 16th. Basically, for the past few weeks I have been involved in the statistics analysis for both of my gene projects using the SPSS software. SPSS is the abbreviation used for Statistical Package for the Social Sciences; it provides almost everything and anything you required to perform an analysis.

Common Statistics Perform in SPSS
In SPSS, it comprises of multiple statistics tests, most of them are covered in year 1 math stats module. Two of the more commonly features used are the descriptive statistics and the bivariate/multivariate statistics.

Descriptive statistics
The most fundamental and frequent statistic feature used. It summarized the samples’ results and portrays the tabulated results in forms of graphs or tables. Thence it is mainly used for quick and basic analysis. Here are some of the statistics tests used in descriptive statistics:
Frequencies
A simple measurement used to computes/determine the mean, median, modes and SD of the results (variables).

Cross tabulation
A cross tab provide information on 2 or more variables’ distribution consecutively and is usually presents a table format. Therefore, a cross tab is different from a frequency test. Some of the statistics tests used abide by with cross tab are chi-square (which test for clinical significant in variables), contingency coefficient (which test for strong interaction between variables) and phi coefficient (which test for degree of interaction between variables).

Bivariate/Multivariate Statistics
As the name implied, this feature is only suitable for 2 variables. Any statistics involving more variables would be under the multivariate statistics. Similarly here is a list of the some of the statistics tests used in bivariate statistics:

t-test
Commonly used when the sample size is minute or when the standard deviation is unknown. It is used to test if a null hypothesis is true or false (accept or reject). For example, if null hypothesis used is: mean of A = mean of B (where both the means of the variables is equal). After a series of calculation if t < a =" mean">

ANOVA
An ANOVA test compares the variables’ mean with their variances. Unlike t-test, ANOVA used variances instead of t value and also assess on more than two variables together.

So how and wat statistics should be applied to different data analysis?
The answer would depend on wat type of “story “ you wish to express in your research. For instance, if you wish to find out the relationship of drug X on blood glucose level. Then a linear regression approach would be an ideal.

A linear regression is a technique used to determine the relationship of dependent variables with independent variables. In this case, blood glucose level is your dependent variable while the dosage of drug X would be the independent variables.
Once you identify your variables you can start analyzed using both the descriptive statistics and the bivariate/multivariate statistics.

Another technique used for data analysis is called the factors analysis. This approach look into dependent variables, meanwhile, indirectly identifies the independent variables within. For example, by looking at blood glucose level (dependent variables), other independent variable such as concentration of drug X would be determined.

Other technique includes K-means cluster analysis, Hierarchical cluster analysis and Ordinal regression.

Lost in this statistics info?? Don’t worry maybe these definitions can help out.
Dependent variables
A dependent variable is an outcome, a prediction that can be influenced by the independent variable. Usually is something, which cannot be control.

Independent variables
An independent variable is something, which you may control or predict in experiment.

Means
Mean equal average

Null hypothesis
A null hypothesis is a presumed statement before statistics analysis

Data analysis
For SPSS to analysis a data/ samples results, a syntax is often used. Simply type in the code (instructions) you want the SPSS to do and select run. Subsequently, the results will appear in the output file in a graph, table, histogram formats (etc). Based on the tabulated results, interpretations can be made.

That all for this week!! Stay tune to next week SIP sharing!!
If you have any question on my post feel free to leave comment.

Avery (0503292E)
TG02

Week 15!

2007-10-01T23:22:00.000-07:00

Hello guys. My turn again! =) After 2 entries about protein expression (Immunohistochemistry/Immunofluorescence), this week, I will focus on GENE expression.

Anyway, I am currently extracting RNA from Formalin-fixed, Paraffin-embedded blocks. Hence, I shall talk about this process today yup!

Reason for extraction/isolation of RNA : Using this RNA extracted from the blocks, I will carry out Reverse-Transcription PCR to reverse-transcribe the mRNA I have gotten into cDNA and then carry out Real-time PCR to look at the genetic expression of a particular gene I am interested in. For more info on Real-time PCR, do refer to my group member, Avery’s blog entry (July 22, 07) as I believe she had written a very detailed entry about this technique =))

RNA isolation must be carried out over 2 days.
DAY 1 :
(Sectioning)

Using the microtome, I will section about 45um of tissue sections into a eppendorf tube.

(Deparaffinissation)

Add 800uL of xylene into the tube and mix.
Significance: Since I am using FFPE blocks, I will first have to remove the wax from the section .

Centrifuge for 2 mins at maximum speed and discard supernatant. Repeat these 2 steps again.

Add 800uL of Abs Ethanol into the tube and mix.
Significance: To remove the xylene from the sections completely. It is also the start of rehydration.

Centrifuge for 2 mins at maximum speed and discard supernatant. Repeat these 2 steps again.

Add 800uL of 70% Ethanol into the tube, mix and repeat centrifuge.

Blot the tube briefly onto a paper towel to get rid of the ethanol residues.

Dry the tissue pellet for 10mins at 55ºC

(Overnight Proteinase K Digestion Incubation)

Add 100uL of Tissue Lysis Buffer, 16uL 10% SDS and 40uL proteinase K into the dried tubes and vortex briefly before incubating the tube overnight at 55ºC.
Significance: This is to disrupt the protein structures in order to get the mRNA we want

DAY 2:

(RNA isolation)

Add 325uL of binding buffer and abs ethanol to the tube and pipette to mix.

Significance: Binding buffer will bind to the nucleic acids we want and allow the contaminants to flow through the filter and into the collection for discard.

Combine the filter tube and collection tube and pipette the lysate into the upper reservoir

Centrifuge for 30 secs at 8000rpm and discard the flowthrough.

Add 500uL of Wash Buffer I to the upper reservoir. Centrifuge for 15secs @ 8000rpm. Discard flowthrough.

Add 500uL of Wash Buffer II and repeat centrifuge.

Add 300uL of Wash Buffer II and repeat centrifuge.

Significance: The nucleic acids will bound to the chaotropic salts specifically to the surface of the glass fibers pre-packed in the filter tube while all other contaminants (salts, proteins and other cellular contaminants) will be washed off the column.

Centrifuge the High Pure filter for 2 mins at max speed.

Place the High Pure filter tube into a fresh 1.5ml reaction tube, add 90uL Elution Buffer and centrifuge for 1 min @ 8000rpm before collecting the flowthrough.

Add 10uL DNase Incubation Buffer and 1.0uL DNase I to the eluate and mix. Incubate for 45mins at 37 ºC

Significance: DNase I will help to remove any residual DNA since all we want is the RNA from the blocks.

Add 20uL Tissue Lysis Buffer, 18uL 10% SDS and 40uL Proteinase K to the eluate. Vortex briefly and incubate for 1 hour at 55ºC

Significance: A second incubation step with Proteinase K is to improve on the quality of the RNA as well as to ensure that all protein structures are disrupted.

Repeat the steps involved for RNA ISOLATION until the part in which the High Pure filter is centrifuged at max speed for 2 mins.

This time round, add only 50uL of Elution Buffer and centrifuge for 1 min@ 8000rpm.

Using the NanoDrop spectrophotometer, we can then check the RNA concentration of the 50uL eluate we obtained from our RNA extraction! =)

All the reagents/buffer were from this kit known as High Pure RNA Paraffin Kit from Roche, Switzerland. Therefore, i do not know the content of some of the reagents such as the Wash Buffer I and II which is why i cannot specifically tell you guys what each and every one of the reagents does ya. Hope this is alright with you guys. But i have tried to explain as many steps as possible so the whole idea behind this RNA isolation process should be pretty clear =)

Difficulties Met During RNA extraction from Formalin-fixed Paraffin-embedded blocks:

(1) During sectioning, the tissue blocks are constantly exposed to the existence of RNase in the surrounding. Hence, the RNA in these blocks might risk getting destroyed during the sectioning process and affecting the total concentration of the RNA after the RNA extraction is done by the end of Day 2.
(2) The process of fixing the tissue sample and embedding it in paraffin has caused severe degradation of the RNA. Therefore, it’s more difficult to isolate good quality RNA from FFPE tissues.
(3) The “older” FFPE blocks tend to give poorer RNA concentration as compared to the tissues that were fixed only recently.

Ways to overcome these difficulties:

(1) Use RNase Zap to help reduce the amount of RNase that could be present in the surrounding (microtome/gloves). If talking is necessary during the isolation process, one could wear facemask so as to prevent the RNase in the saliva from degrading the samples.
(2) Since it’s more difficult to isolate RNA from FFPE tissues, more sections could be collected in several tubes and pooled together so as to maximize the concentration of RNA to be collected after 2 days of extraction.
(3) If given a choice, choose tissue blocks that were fixed and embedded recently (within 5 yrs) because studies have found that RNA in the blocks that were more than 10 year-old degrades significantly.

Kangting

0503331A

TG01

Week 14- Confocal Microscopy!

2007-09-29T11:35:00.000-07:00

Hey..this week’s my turn, and I have interesting PICS to share from my research work.

As I have mentioned before, my project is basically delivering DNAs into cancer cells using synthetic carriers such as polymers for future gene therapies. Hence, I’m doing the last part of my project which is ‘localisation of polymer-DNA complexes’ in the cells.

For the localization studies, my main aim is to determine whether my polymer goes into the nucleus. Hence I have to label my polymer, nucleus and DNA. However, since my DNA labeling kit has not arrived, and I want to train myself using the confocal microscope, I started my experiment first using my labeled polymer and nuclear labeling.

The cell line I chose is HepG2, it is a human hepatocellular carcinoma cells, mainly because it does not detached easily from the tissue culture plate. I label my polymer with FITC, and to stain the nuclei, I used Hoechst dye that is specially used to stain ‘live’ cells.

Why use CLSM?

In order to view my cells, Confocal laser scanning microscopy(CLSM) is used. The CLSM used is LSM 5 LIVE, the latest edition for imaging of living cells from ZEISS technology. CLSM uses laser to excite the fluorochromes, unlike fluorescence microscope(FM) which uses fluorescence light.

1)CLSM allows 3D view of the sample, hence if there is an overlapping of different fluorescent signals, only CLSM can help differenciate them.

2) CLSM allows viewing of distinct and sharp images by removing out-of-focus light, unlike FM that usually gives blurry images and some signals cannot be viewed distinctly.

If you wanna know the principle of CLSM and FM, just give me a comment, as it is more like ‘physics’, afraid some of you might not understand.

Ok back to my experiment, I prepared 4 different samples at different time interval: 1, 2, 3 and 4.5 hr, these time intervals are chosen after reading some science papers and also FITC ‘photobleaches’ easily, hence I have to prepare samples at different time intervals. The polymer-DNA complexes are given(transfected) to the cells at 0 hr, while the nuclei are stained with Hoechst 40 mins before viewing under CLSM, and the cells are washed 5 times with colourless DMEM medium before viewing to prevent auto-fluorescence. Hence, that is why i used HepG2, because of the washing steps.:)

One important precaution: sample preparations must be done in the dark, once exposed to light, the fluoresce intensity can greatly decrease, especially for FITC.

GREEN(FITC): polymer ( note: DNA is still bound to the polymer, but cannot be seen as it is not labeled)

BLUE(HOECHST): nucleus

The following pictures are a bit blurry because I magnify them 8-12 times from the original one.

1 hr after Transfection: on the cell membrane

Q: Why do the polymers 'stick' onto the membrane?

A: My polymers are cationic(+vely charged), and the cell membrane has proteoglycans(-vely charged), hence the ATTRACTION!

2.5 hr after Transfection: in the cytoplasm

Q: How does my polymer enters the cytoplasm?

A: Based on other science papers, it is known that polymer-DNA complexes enter the cells via endocytosis. However the actual mechanism(type of endocytosis) is still a BIG question, and no one knows.

4.5 hr after transfection: in the nucleus!!

Q: How can the polymers(size: 100-200nm) go into the nucleus, when nuclear pores only allow molecules of less than 10 nm to pass through?

A: This is also a another BIG question that many scientists are still finding. However based on latest papers, it is known that cationic polymers do interact with the anionic phospholipids on the cell membrane and eventually coat the polymer-DNA complexes. These coated complexes then fuse with the nulear envelope, and release the complexes.

Anyway, frankly, i hate 'Live' confocal studies, because it is very tedious, and u will spend the entire day on it, plus TIME is a GREAT factor. Also usually, there will be cells that do not show similiar pattern like others under the microcope, hence, I have to search and search. :(
But still, research is FUN! :))))

Nisha
TGo2
0503254E

Week 13 Sharing of experiences in histopathological laboratory

2007-09-21T04:23:00.000-07:00

In tissue processing room

Whole mount preparation
-Completely cross-sections of a pathological specimen
-Usually too large for the ordinary cassette and require individual handling prior to machine processing
-Example: whole mount sections of prostate

Observations of prostate trimming by pathologists
-Material: Prostate cutting tool
-Whole mount is necessary as tumour is located in prostate and can only be seen under microscope.
-For whole mount processing, the timing of fixation will be extended for 41/2
hours (total 211/2 hours) to ensure tissue is thoroughly fixed.
-The dehydration steps will also be extended to ensure proper dehydration due to the size of the tissue.
-Other tissues will not be processed together with whole mount prostate as over fixation and dehydration of smaller pieces will cause the tissues to be harder.

Microtomy

1. Microtome safety (Safe Operating Procedure)
2. Handle blades very carefully when installing or removing. Follow the manufacturer’s guidelines explicitly.
3. Tungsten-Carbide knives can put through your shoes, if dropped. Be careful where your feet are positioned when installing or removing blades.
4. Store blades in a covered container that has guides to hold the blades rigid.
5. Never leave blades on countertops. Lacerations can occur when reaching across the countertop and inadvertently contacting on unprotected table.
6. When setting up the microtome, position the sample first, and then put in the blade. Never the other way around.
7. When applying the brake, ensure that it is tight. Most accidents occur when the brake slips and the operator’s hand is drawn into the blade.
8. When leaving the microtome, even for a short time, ensure that the blade guard is in place.
9. When preparing a paraffin sample for the microtome, remember to clamp the sample down tight. The movement is allowed by a loose clamp increase your risks of cut.
10. Use forceps or brushes during retrieving and transferring slides and ribbons from the blade, thereby keeping your hands free from the moving parts of the microtome.

Common faults encountered in microtomy and remedies

Causes / Remedies

Ribbon fails to form
1. Paraffin too hard / 1. Use softer wax i.e. lower melting
2. Blade too blunt point / 2. Change to a new blade edge
3. The tilt is too great / 3. Tilt the blade less

Crooked ribbons
1. Edge of block is trimmed not parallel to blade. / 1. Re-trim the block.
2. Irregularities in blade edge / 2. Try another part of the blade

Sections compressed, wrinkled and jammed together
1. Blade too blunt / 1. Change to a new blade edge.
2. Paraffin block is warm / 2. Cool the block on cryoplate
3. Blade tilt is too slight, therefore / 3. Increase the tilt
the cutting facet rubs over the block
4. Blade edge gummed with wax / 4. Wipe blade with xylene

Sections crumble and specimens fall out, mushy and soft
1. Incomplete dehydration / 1. Re-hydrate
2. Improper embedding / 2. Re-embed

Tissues turn hard and cut like stone
1. After clearing, the specimen was / 1. Nothing can be done except repeat
accidentally left to dry in air with fresh specimen
2. Paraffin bath is too hot and burns the tissue / 2. Decrease the temperature of parrafin
bath

Split ribbon or lengthwise scratches in ribbon
1. Nicks in the blade / 1. Use a different part of the blade.
2. The tilt of the knife is too great / 2. Decrease the tilt
3. Blade edge dirty / 3. Wipe blade edge with xylene
4. Hard particles (calcified materials) in the block / 4. Do surface decalcification
5. Crystal from fixative e.g. Mercuric chloride / 5. Treat tissues to remove crystals

Undulations in the surface of the section
1. The blade is not tightened / 1. Tighten all screws
2. The title of the blade is too great / 2. Decrease tilt to prevent vibration
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4. Blade edge is dull or rounded / 4. Change to new blade edge

Sections of varying thickness and skipping of sections
1. Blade not tilted enough to clear facet, / 1. Adjust the tilt
or too much and tissue is compressed until
the inevitable expansion gives a thick section
2. The blade and specimen holder not tightened / 2. Tighten all screws
3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex
4.Cutting stroke is not regular or interrupted / 4. Maintain regular cutting stroke

Sections full of fine lines running across
1. Blade edge consist of fine serration / 1. Change to a new blade edge
2. Cutting edge is dirty / 2. Clean blade edge

Sections full of folds
1. Water bath is too hot or too cold / 1. Adjust temperature to 50 degree
Celsius
2.Blade tilt is too slight so cutting facet / 2. Increase the hilt
rubs over the block.
3. Paraffin block is warm / 3. Cool block on cryoplate

Alright i shall stop here then. :)

Ang Xiao Si Sharon
TG02
0503219H

Week 12 Attachment Sharing

2007-09-16T05:26:00.000-07:00

Hey guys! 12 weeks have passed. I have started with a second project that covers expression proteomics of a clinical isolate of Stenotrophomonas maltophilia, a nosocomial infectious agent. Expression proteomics relates to studying the entire secretory profile of the bacteria. In my case, i will be precipitating the secretory proteins from S. maltophilia grown at both 28˚C and 37˚C.

Methods
1) Isolate is streaked on an LB agar plate and incubated at 37˚C for 24 hours
2) A colony will be inoculated into 20mL of LB broth and incubated at 28˚C for 24 hours
3) 5x10^7 cells will be inoculated into 12 tubes in which 6 will be incubated at 28˚C and the
other 6 will be incubated at 37˚C
4) Secretory proteins in the supernatant after centrifugation will be transfered to teflon tubes
5) Equal volumes of 40% TCA in Acetone as the supernatant will be added to precipitate the
proteins
6) Tubes will be incubated at 4˚C for 1 hour and inverted every 10 mins
7) Tubes are then centrifuged at 16,000xg for 1 hour at 4˚C
8) Keeping an eye on the protein pellet, supernatant is decanted
9)Remaining supernatant is aspirated out and 250uL of pre-chilled Acetone is added
10) Acetone is used to wash the proteins
11) Washed protein is transfered to a fresh Eppendorf tube and centrifuged
12) Supernatant is aspirated and pellet is allowed to air-dry
13) 200uL of Rehydration Buffer is used to rehydrate the protein pellet
14) 2uL of Protease inhibitor was added to prevent denaturation of proteins
15) Bradford Assay is performed to determine the final protein concentration
16) If there is sufficient protein sample, a 2-Dimensional gel can be run
17) Spots containing significant proteins can be identified and excised
18) Protein spots are then analyzed through the MALDI TOF/TOF
19) Peptides can then b identified through the Mascot Search program

Chemicals
That was a brief itinery of what is carried out in my experiments daily. A single experiment can last for up to 2 weeks. Now i'll explain about certain chemicals that are required for protein precipitation.

Acetone is used for the washing of protein pellets after precipitating with 40% TCA in Acetone. Acetone is the simplest and most important of the ketones. It is a polar organic solvent and therefore dissolves a wide variety of substances. It has low chemical reactivity. These traits, and its relatively low cost, make it the solvent of choice for many processes. About 25% of the acetone produced is used directly as a solvent. Acetone is also used as a drying agent, due to the readiness with which it binds to water, and its volatility. Also, by washing with Acetone, any impurities that have accumulated in the process is removed from the proteins.

Since Acetone can act as a drying agent, the protein pellet will be rid of most liquids thus Rehydration Buffer is required to rehydrate the proteins. The Protease Inhibitor is used to prevent any proteases from acting on the precipitated proteins. Proteases may still be present because they ar enzymes which are also proteins.

Aseptic techniques
When working with proteins to perform analysis, it is important to not come into contact with the samples since it can contribute to the quality and quantity of proteins analyzed. For example, if we do not wear gloves and come into contact with the sample, keratin may be found abundantly in our protein sample. If we talk into our tubes during air-drying, enzymes may be added to our samples.

Conclusion
Thus, i conclude by saying that even though research may seem easy to some and boring to others, i have to say that they are very wrong. I have enjoyed my 12 weeks at the research centre so far through all the ups and downs and all the "failed" experiments. It is inevitable not to get good results but they ARE results so we learn from them. This is the end of my second entry after 12 weeks. Hope to blog in 6 weeks time with more interesting facts to share. See you guys at the next Campus Discussion!

Johanna
0503309G
TG02

Week 11 Attachment Sharing

2007-09-09T05:32:00.000-07:00

Heya guys....how are all of u doing......???11 weeks have passed.......9 more weeks to go...dunnoe if that's good news or bad news....8-?......ahkahkahk....aniwei this time round....i would be blogging about something all of us are very familiar with.......PCR!!!.......

Title: PCR of subjects' DNA (24 for Chinese, Malays and Indians each)

Purpose: To make a lot of copies of the specific DNA fragment that needs to be analysed.

Materials and Methods:

1. Prepare master mix for 75 samples containing the following components:

2. Pipette 8ul of the master mix into each well of the 96-well PCR plate.

*those boxes coloured are filled with the 8ul master mix.

3. Pipette 2ul of the DNA into the wells in the following order:

4. Cover the plate with silicon mat

5. Spin down the plate at 200G for 20 sec

6. Put the plate in the PCR machines. The standard conditions for PCR are:

7. After PCR has finished, spin down the plates at 200G for 20 sec

That's all for now...hope u guys enjoy the last few weeks of your SIP...all da best guys!!!!:)

Nur Zahirah

0503165C

TG02

Week 10 Attachment Sharing

2007-09-02T10:28:00.000-07:00

Hi guys!! It is my turn to blog again! For the past few weeks I have been handling 2 projects on allele discrimination. As mentioned previously in my blog, I used allelic discrimination kit to identify the different alleles present in the samples provided. However, there is another method to differential the allele, namely the Enzyme-based method. This method involved a lot of molecular biology principle and is quite time consuming too.

Despite that the duration for this method is rather long, the results produced is much more easier to analyze and interpret. Nevertheless, this method is not suitable for most of allele discrimination. It can only be used if the polymorphism of the allele can be ‘coincidently’ digested by any specific restriction enzymes. In my case, it happens that the alleles we emphasized are different by a nucleotide and can be digested by a particular enzyme.

Recipe for Enzyme-based methods

1.First and foremost, the DNA samples are amplified by using the PCR thermocycler and specific primers (both forward and reverse) close to the target gene.
2.The DNA samples then undergoes restriction digestion performed by a specific restriction enzyme (it is very important that the restriction enzymes cutting site matches with the polymorphism).
3. Digested samples are run in gel for separation of alleles
4.Stained the gels in EtBr (Recommendation: add EtBr when casting gels, it is a lot better!!)
5. The gels are then detected under UV light.

Interpretation of results:

Since the restriction enzymes is able to use the polymorphism as it cutting site, only DNA samples that comprised of the allele will be digested. For example if allele y contains the restriction enzymes cutting site (contains the target nucleotide), restriction enzymes will be able to cut the allele into two fragments. In contrast, if it is allele Y then there will be no cutting site for the enzymes to act on. As a result, allele y will appear below allele Y after electrophoresis. This is because allele y has been digested into smaller fragments and hence will drift faster than allele Y in the gels. Then again if the DNA samples is a heterozygous, there will be a double bands. Since only one of the chromosomes contains the cutting site, this amount of chromosomes digested will also be half while the rest remain uncut. Hence, a double band will appear as half of the digested fragment (digested one) drift faster than another half (undigested).

Other methods for allele discrimination:

Dynamic allele-specific hybridization (DASH)
Basically this method worked by measuring the difference in melting temperate in the DNA samples. Unlike the other methods, DASH used biotinylated primer with a bead attached to it. The biotinylated primer with the bead is then extend the DNA samples is amplified.
The amplified DNA samples is then incubate in streptavidin column. NaOH is then added to wash away the unbiotinylated DNA. This is because the unbiotinylated DNA does not have the bead joined it and attached to the column. An fluoresces allele specific oligonucleotide is then added to form hybrid with the amplicon. Last but not least, heat is given to determine the Tm of the DNA samples. If there is present of single nucleotide polymorphism the Tm will be lower.

PCR-based methods

In this method, it used two pairs of PCR primers. Each of these primers are specific to their respective alleles.The primer consist of the single nucleotide polymorphorism.Since each pair of primers anneal specifically to their allele. When undergoes PCR process only either one of the primers will bind to the target DNA and amplified.

That all for the week!! Hope your guys have fun and feel free to give comment!!

TG02

Avery, May Lee ( 0503292E)

WEEK 9

2007-08-20T19:43:00.000-07:00

WOWWWW. Time flies. It's the 9th week =)) Hope u guys are coping well.

Anyway, this week, i will be talking about yet another skill that i picked up during my SIP - IMMUNOFLUORESCENCE. I believe just last week, Ye Tun has talked about this technique (http://sevenseven77.blogspot.com/) that he is using for viral identification. In my case, I am using it to show the localisation of a particular protein in the cell (whether it's in the nucleus or cytoplasm). It 's something really similar to Immunohistochemistry which i mentioned in WEEK 3 so i will skip explaining several steps ok? =)

Aim of test: I am using TWO primary Ab and of course 2 secondary Ab labelled with fluorescence substrate. The 2 fluorescence substrate i am using are FITC (giving the apple green fluorescence signal) and the TRITC (giving the red fluorescence signal) on one single section. Hence, when viewed under the fluorescence microscope, you should be able to see both colors!

Step 1 - 10 : Pls refer to the immunohistochemistry steps i wrote for WEEK 3

Step 11: Secondary Ab labelled with FITC was added. Since this 1st primary Ab that i am using is raised in mouse, my secondary Ab is an anti-mouse solution yup! Addition of Secondary Ab must be done in the dark so as to prevent the exposure of the fluorescence substrate and the slides was placed in a dish and wrapped in aluminium foil for the same purpose.

Step 12: Washing carried out

Step 13: This time round, the 2nd different kind of primary Ab was added

Step 14: Secondary Ab labelled with TRITC was added. Since this 2nd primary Ab that i am using is raised in rabbit, my secondary Ab is an anti-rabbit solution yup! Addition of Secondary Ab must be done in the dark so as to prevent the exposure of the fluorescence substrate and the slides was placed in a dish and wrapped in aluminium foil for the same purpose.

Significance: It is very important that you choose the primary Abs raised in different animals should you be using 2 antibodies on one single slide. This is because if both my 1st and 2nd primary Abs are raised in mouse, when secondary Ab is added, it will bind to BOTH the primary Abs instead of just one. Then, when you obtain your results, you wouldn't know if the Secondary Ab is binding specifically to its specific primary Ab.

Step 15: Washing was carried out once again.

Step 16: DAPI was added as a counter stain

Significance: DAPI is able to allow the nucleus of the cells to emit blue fluorescence signal. Hence, when viewed under the microscope, we would be able to know which cells are not emitting the fluoroscence at all (by only showing the blue DAPI fluorescence without the red/green fluorescence signal), hence showing no expression of that particular protein of interest.

Step 17: Washing done again

Step 18: Mounting medium was added onto the slide and cover-slipped.

Step 19: View under the fluorescence microscope! =)

As you can see from this pic, there are both the green and red fluorescence. In area where there is a slight tinge of yellow/orange, it is most likely where the 2 different proteins (Remember we are using 2 primary Abs?) co-localize. However, we need the confocal microscope in order to confirm if these 2 proteins are located in the exact same area.

Hope u guys understand my entry. haha. n take care =))

Chen Kangting
0503331A
TG02

Week 8 Attachment Sharing

2007-08-17T22:31:00.000-07:00

Hey wait! Don’t switch to another blog. One of my colleagues gave me a unique and interesting exam question given in a university. If one day you become a lecturer, you should set such questions. Go read the 1st comment for this post!

Ok..now the boring part..

This few weeks I have learnt quite a lot of new stuff like flow cytometry analysis, plasmid purification, gel retardation assay and also confocal fluorescence microscope(still learning). However, I will focus on flow cytometry in this week post because I think it is more interesting.

Introduction

In this experiment, cancer cells are tranfected with polymer-DNA complexes. Since naked DNA/plasmid cannot be efficiently delivered into the nucleus of the cells, my lab designs carriers such as cationic polymers to deliver the DNA into the cells. In this case, the DNA is an EGFP encoded plasmid, meaning, cells that receive the plasmid are able to express the green fluorescence protein(GFP). Such DNAs are also termed reporter genes. To measure how good the carriers are in delivering the plasmid, we measure the level of gene expression.

Purpose of flow cytometry in this experiment

Flow cytometer is used to measure the gene expression. Cells tranfected with the EGFP plasmid will fluoresce green when exposed to blue light. The unique factor of flow cytometer is that it can measure the fluorescence per cell, hence you can know how many cells are expressing the gene. Unlike normal spectrophotometers which measure the transmission of light in a bulk of sample.

Flow cytometers can measure more that 1 type of fluorescence simultaneously per cell and also measure different types of cell. However in this experiment, the cell type used is HEK 293(human embryonic kidney cells) and the fluorochrome is green fluorescence protein(GFP). The brand of the flow cytometer used is BD LSR II( 3-laser FACS analyzer).

Before, using the machine, cells must be prepared for analysis. Single cell suspensions must be obtained. Though the process is simple, but it is very tedious and time consuming. I don’t see the significance of explaining individual steps, so if you are interested, just send me a comment.

I usually run 3 replicates for each condition(different concentration of polymer-DNA complexes added into cells). In this experiment, I have 10 condition, hence, I will have 10x3=30 samples. The whole process can take a day! I’m always super exhausted at the end of the day.

Principle of flow cytometry

Image 1: taken from http://biology.berkeley.edu/crl/flow_cytometry_basic.html

When measuring, cells will flow into a stream of fluid in a flow chamber, allowing only single cells to pass due to the reduced diameter. Lasers are often used as light source in flow cytometry. In this case, argon ion laser is used to excite the GFP cells at 488nm. The corresponding fluorescence signals are picked up by optical detectors at 503-530nm.
The signals are then ‘processed’ further, but it is so technical, so no need to explain as the machine does everything. =))

Data presentation

This is the most important part. A computer is connected to the flow cytometer to analyze the signals. It can be presented in many ways, but I only use two, histograms and dot plots.

1) Dot plots( 2 dimension)

Image 2

y-axis: SSC-A(Side SCatter): parameter that relates to the density/granulariy of the cell.
X-axis: FSC-A (Forward SCatter): parameter that relates to the size of the cell.

Both axes allow us to create a 2 dimensional plot.

Dot plots are often used to measure cell size and density. Based on both axes, it will position the cell in a form of ‘dot’. Each dot represents 1 cell. Only cells(dots) that are in the scatter gate are analyzed, while the others are ignored. The scatter gate is created by the user(me =)). Hence, it can be shifted to the right or left, or even make it larger. So how do I decide? I use controls and all my other samples are based on that.

Why do I need controls?

Reason: To properly set the conditions for flow cytometry, negative (untreated cells: no GFP) and positive controls are required for each cell type and for each fluorescence dye. Different cell types auto-fluoresce at different intensities, so a negative control for one cell type might appear positive for another cell type. In addition, if one cell type is significantly larger or more complex than the other, their forward- and side-scatter settings will differ.

In real life, dot plots are often used in hematology to distinguish different lineages of blood cells, as different type of cells will appear at different parts of the plot based on their density and size.

2) Histogram(single dimension)

Image 3

y-axis: count: no. of cells
x-axis: GFP-A: relative fluorescence intensity

This is the simplest way to present the data. Peaks that are in the right ‘box’ are GFP positive, while those in the left are non-GFP cells. Again, I must have negative and positive controls to ‘gate’ the peaks.

Finally, The flow cytometer will automatically tell me how many GFP positive cells based on the data analysed and the scatter gate I chose.

Image 4: Interpretation of data

Note: Image 2-4 are posted with permission.

Till here then.

Nisha

0503254E

TG02

Sip sharing..cytology!

2007-08-11T11:12:00.000-07:00

Hey people! I’m now attached to a routine cytology laboratory. My tasks assigned are: mount stained slides manually, sort slides, receiving/collection of specimens (both gynae & non-gynae), processed urine and sputum samples. So this time, I will blog on the procedure for sputum processing and Papanicolaou staining.

Sputum testing is to differentiate the nucleus and cytoplasm of the cells to detect malignancy by comparing the Nucleus: Cytoplasm ratio (N:C ratio).

Procedure for sputum processing
1. Receive sputum sample
2. heck particulars of patient and nature/number of specimen
3. Assign a doctor to the patient and stamp date/time received and initialized by technician
4. Label all containers containing sample with pre-printed labels
5. Initial of technician performing sputum sample and the condition of sputum sample
6. Label the specimen lab number on the frosted end of the four clean glass slides.
7. Label on one of the slide the name of the special stain, if requested example Ziehl Neelsen
8. Pick the selected material with a Pasteur pipette and flush them onto the surface of the labeled glass slide
9. Holding a slide on one hand, pick up another slide to pull the material evenly over their surface to make smear. Avoid leaving rough or raised areas that may cause cover-slipping problems.
10. Used Pasteur pipette are being disinfected in sodium hypochlorite.
11. Place slide immediately into container containing 95% ethanol. 12.Separate out slides for special staining.
13. Additional slides are prepared if future request for special stains are made.
14. Load slides into autostainer using slide hold (rack)
15. Stain with Papnicolaou stain
16. Mount with 24 by 50mm cover slips and Depex manually
17. Place in oven for 10 to 15 minutes to let Depex dried up
18. Slides are ready to be observed under microscope

Label with specimen lab number and name of specimen
A drop of sputum sample

Making a smear using the ‘touch and pull’ method’

A smear done

0.5% Sodium Hypochlorite (prepare and use only fresh solution) is a disinfectant. It is active against bacteria, spores, fungi, viruses including HIV and HB. Concentration is 0.50%.
Used for specimen disposal, contaminated waste, materials spillage and non-metal equipments. The contact time must be at least 30 minutes for optimal effectiveness.

Routine Papanicolaou Staining
Papanicolaou method is a polychrome staining reaction (staining the cytoplasm of different cell types different colors) designed to exhibit differences in cellular morphology, maturity and metabolic activity
Intact cells in a cytologic smear tend to overlap and some appear in three dimensional configurations, the greatest value of papnicolaou staining method are the resultant transparency of the cells and clear definition of nuclear detail
Used for gynaecologic and non-gynaecologic specimens

Principles
1.Fixation
2.Nuclear Staining
3.Cytoplasmic staining
4.Clearing

A series of graded percentage of alcohol (80%-70%-50%) to hydrate the cells gradually before immersion in the aqueous haematoxylin solution
After approximately two minutes in haematoxylin the cells are then dehydrated (70%-80%-95%) prior to immersing in the alcoholic counter stains (Orange G and Eosin Azure)
Following the two cytoplasmic stains, the slides are then rinsed in alcohol

Fixation
To fix/preserve the morphological details of the cell in as perfect a condition as possible

Nuclear staining
Regressive method: The nuclei are overstained with unacidified haematoxylin; excess stain is then removed with dilute hydrochloric acid. The hydrochloric acid must be removed by a bath of running water.
Progressive method: To stain for the desired color intensity. This method eliminates decolorizing with hydrochorite acid and the need for subsequent running in a water bath. Recommend for non-gyanecological cell samples because they do not adhere to slides as well as those from the female genital tract. To cause the colour of the stain to change from red to blue, the slides may be ‘blued’ with dilute solutions of ammonium hydroxide (NH4OH, Ph 8.0-8.5), lithium carbonate ( Li2CO3, pH 8.0-8.5) or Scott’s tap water substitute (pH8.2).

Cytoplasmic staining:
After the nuclear staining, the cells are dehydrated through rinses of 95% alcohol. This dehydration prepares the cells for the two alcohol stains. First, the OG-6 stain, the slides will be in the alcohol for 1 minute. If there is any keratin in the cytoplasm of these cells, the OG-6 will stain it a brilliant orange. Following the OG-6 stain, the cells are rinsed in two 95% alcohol baths. They are then immersed in the modified EA stain for 2 minutes. The modified EA is a combination of these two stains : First is the eosin, which stains the cytoplasm of mature squamous cells, nucleoli and cilia. Second, is light green, which stains the cytoplasm of parabasal and immediate squamous and columnar cells.

Clearing:
Following the EA stain, the cells are then taken through two 95% alcohol rinses. These high concentrations of alcohol help to provide a clearer view through areas of overlapping cells
Next, the cells are taken through 10% alcohol rinses for final dehydration
Upon complete dehydration, the cells are placed in the two to four rinses of xylene where they remain until the coverslipped. The xylene will carry light rays from the microscope in the same way that the cell will, thus, making the cells transparent.

Note: Pictures are taken with the permission from my supervisor.

Yup so thats all :) do feel free to ask any questions..

Sharon Ang

0503219H

Tg02

WEEK 6 ATTACHMENT SHARING

2007-08-05T03:42:00.000-07:00

Hey guys, Johanna here. I am currently working in a research centre that deals with a variety of bacterial work. My research project focuses on protein work of Stenotrophomonas Maltophilia, a bacteria that has been known to cause noscomial infections in hospitals.

My Attachment

My SIP supervisor required us to perform a series of experiments to get a feel of how to run certain methods of bacterial identification tests based on the proteins we have extracted from the bacteria.

Our first SIP assignment was to perform Chloroform Shock on the bacteria. Choloroform shock is a method of extracting the cell surface membrane proteins that would most probably have cell adhesion properties. In the clinical context, this would probably mean that the bacteria's cell surface proteins actually help in adhering to surfaces like hospital equipment, furniture, patient's skin etc. Thus by extracting these proteins, we can carry out studies on these proteins to determine their clinical significance and also provide a basis for further scientific studies on how to fight these rapidly spreading microbes in hospitals.

The chloroform shock method is suppose to be specific to the extraction of cell surface membrane proteins. My group was suppose to determine if it is actually specific and does not cause cell lysis. Cell lysis would affect the experiment because the intracellular proteins would also be released, thus the proteins would not be purely cell surface membrane proteins. We ran the experiment about 6 times before we succeeded in proving that when using chloroform shock, mainly cell surface membrane proteins were extracted. Although there was cell lysis, it was not substantial enough to affect the results of the main experiment.

Our second SIP assignment was to standardize the protocol for the extraction of secretory proteins of the bacteria, S. Maltophilia. The secretory proteins are also clinically significant as these proteins would facilitate in the survival of these bacteria. This is because, different proteins should be secreted in the different clinical environments mainly at different temperatures. However, our SIP supervisor gave us two different clinical isolates, mainly the clinical isolate and environmental isolate. We were suppose to meet a series of objectives - to standardize the inoculum, to determine the cell density of the bacteria after 24hrs of growth and to correlate between the cell density with the number of cells. I will elaborate more on this experiment.

Before performing each experiment, we were suppose to plan out our protocol such that we would not collide with other people's experiments. Also, by planning our protocol, we would be sure of our next step. An example of our protocol would be:

10 July 2007 - Streaking

11 July 2007 - Inoculation

12 July 2007 - Cell density, Serial Dilution and Plating
(i) Methods
a) 18 agar plates were incubated in 37 incubator to dry for subsequent use
b) Centrifuge was set pre-cool and BSC was turned on
c) Materials required were swabbed and placed in BSC (Biosafety Cabinet)
d) Eppendorf tubes and agar plates were labelled
e) Tubes from 28 incubator were taken out, tapes removed and caps tightened
f) Tubes were centrifuges at 3,000xg for 20min at 10 degree Celcius
g) Supernatant was cecanted and cell pellet was resuspended in 10mL of LB broth
h) Washing step was repeated twice
i) Supernatant was decanted and cell pellet was resuspended in 20mL of LB broth
j) 1mL of LB broth was aliquoted into a disposable cuvette to tare cell density meter
k) 1mL of sample is then aliquoted into dosposable cuvette and measured
l) Each sample was measured in triplicates
m) Serial dilution was performed for chosen samples
n) The 6th, 7th and 8th dilution samples were plated on LB agar plates in triplicates

After performing those steps, we will incubate the plates for 24 hours before doing a plate count. from the plate count, we can determine if our dilution was performed correctly. If we did it right, there would be an obvious dilutional effect. Then, we can compare the results from the cell density readings and plate count to determine the number of cells in 1 OD (optical density).

When we carried out this experiment the first 4 times, we had a series of contamination cases. We carried out a lot of troubleshooting for the first two times and found out that the micropipette we used was contaminated. The following two tries, we still had contamintaion though it was significantly lesser than the first two and our results were not badly affected. However, to collect solid evidence that the protocol would provide good data, we were suppose to carry out more tests to confirm this.

Reflecting

Besides all the experiments we carried out, we also had to prepare all the materials that we require in the lab for our experiments such as the LB broth, LB agar, PBS used for serial dilution, DI water etc. We also had to keep the labs clean to enable easy assess to equipments and such. We also had to read a lot of protocols and literature reviews on the machinery we were to use such as the Xcise, MALDI TOF/TOF and the soon to come laser scanner for CyDyes.

Therefore, being in a research laboratory really teaches us a lot and makes us think on our feet and how to handle problems by troubleshooting and solving the problem. It also helps us to organize our time and think about others that work in the lab.

That's all i have to say for this post. Any questions don't hesitate to ask OK? See you guys in school soon!!

Johanna
TG02

WEEK 5 ATTACHMENT SHARING

2007-07-29T04:16:00.000-07:00

Heya guys……zahirah here….i’m currently attached to clinical pharmacology lab with michelle…if u guys think that we are in clinical lab..then u guys are super duper wrong..hahaha…well we are actually in a research lab doing studies on the genes of different ethnic group and see how it might affect the pharmacodynamics and kinetics of the treatment…think BPHARM….but then our lab deals with the major techniques in MBIO which is PCR and DNA sequencing…since we are together and to avoid u guys from reading the same thing twice….we will try our best to post up on different things….:)

Title: Running Gel electrohphoresis
Purpose: To determine the optimal temperature of the primers that we are going to use for the research by looking at the number and intensity of the bands.

REFER TO MICHELLE’S BLOG (http://jammys-bms.blogspot.com/) FIRST FOR THE OPTIMIZATION OF PRIMER

Before we run the gel…of course we need 2 make the gel first and it is prepared while waiting for the PCR to finish..save time a bit….soo..here’s the ‘ingredients’ and ‘recipe’ for the gel

For making agarose gel
Materials:
Agarose powder 1X TAE buffer Ethidium bromide Combs Tray
Conical flask Measuring cylinder

Method:
1.Prepare the tray by covering the open ends and pour DI water to measure how
much volume of TAE buffer is needed
-the volume needed is dependant on how thick you want your agarose to be
2.Pour away the DI water and place the tray in the fume hood
3.Calculate the amount of agarose powder needed
-this is calculated using the formula:
% of agarose gel x vol. of TAE buffer = weight of agarose powder
-since in this lab we use 1.5% agarose and the volume of TAE buffer needed is
70ml
thus: 1.5% x 70ml = 1.05g
4.Weigh 1.05g of agarose powder and put it in the flask
5.Measure 70ml of 1X TAE buffer
6.Pour the 1X TAE buffer into the flask and swirl gently
7.Place it in the microwave for 50 sec
8.Once you get a clear solution, bring the flask to the fume hood and swirl for a
few seconds first
9.Add 3.5ul of ethidium bromide into the flask after the solution has cool down a
bit
10.Calculation for vol. of ethidium bromide needed:
Stock conc: 10 000ug/ml
Dilution conc needed: 0.5ug/ml
M1V1=M2V2
0.5ug/ml x 70ml = 10 000ug/ml x V2
V=0.0035ml = 3.5ul
11.Swirl the flask gently to obtained homogeneity
12.Pour the solution into the tray and place the combs

Note: the volume of 1x TAE buffer, agarose powder and ethidium bromide and the number of combs needed is dependant on the size of the tray/agar. For this one, it is for a normal size agar that is able to hold two combs. The thickness of the comb is also dependant on what product we are going to run. For primers, we use a comb with big and thick wells as we are going to load all the PCR products.
For running gel electrophoresis
Materials:
6x loading dye PCR products 100bp ladder 1.5% agarose gel

Method:
1.Place the agarose gel into the gel electrophoresis machine
-ensure there’s enough TAE buffer to cover the gel
-the wells is placed at the negative part of the machine
2.Pipette 2ul of 6x loading dye into each PCR tubes and mix
3.Load the samples to each corresponding well(starting from the 2nd well)
Eg. 1st PCR tube-to the 2nd well
12th PCR tube- to the 13th well
-must ensure that we don’t jumble up the tubes and the loading part because
each tubes represent the product that has undergone different annealing
temperature
4.Pipette 2ul of 100bp ladder to the 1st and last well( after the 13th well)
5.Run the gel at 120V for 15-20 min

++Initially we want to post up the pictures we obtained after gel electrophoresis but when we ask our supervisor for permission, he did not give us a clear cut yes….soo sories guys…but u can refer to your MBIO lecture notes the topic after cloning…the bands we obtained are almost similar to that…

A BIT ON LMQA...
All staff is required to attend safety modules which even include a module on centrifuge safety..at the end of each module, we actually have to sit for a test and even do assignment. All the work done in our lab are pretty manual..except for those that must be done by machines like DNA sequencing, etc...not that advance like nisha's where there's even an automatic pipette filler..our fridge actually have warning that says may contain biological agent because we do store blood from patient's in the same fridge we store our reagents...and there are some labs that have the radioactive symbol(the three triangles) because that particular lab deals with radiation/radioactive materials or they used to deal with it.....

so my posting is till here......shall continue the nxt time i post....wishing everybody all the best for your SIP/MP and enjoy....tata...:)

Week 4 Attachment Sharing

2007-07-22T09:06:00.000-07:00

Greeting to all fellow BMT course mates!! It is time for me to share my experience in SIP with you all. Let begin!!

Basically, I am attached to a research lab, which revolves around gene expression and its relation to certain disease. Concurrently, I am working on a particular gene, which might be related to diabetes. In this research, most of time I am dealing with a lot of MBIO and MCT stuffs, for instance things like PCR, real-time PCR, cell culturing and DNA sequencing.

Introduction to real-time PCR
Real-time PCR also refers to kinetic polymerase chain reaction. It is a unqiue technique commonly used to amplify and at the same time qualify the amount of a specific DNA of choice in the sample. In fact, it detects the amount of target DNA based on the amount of fluorescence release from its probes during the PCR process.

Principles of real-time PCR
Let recall our biochemistry and molecular biology knowledge! As we have learnt in lessons, PCR amplified double- stranded DNA by adding Taq DNA ploymerase, dNTPs, DNA templates, PCR buffer, last but not least the primers (both forward and reverse). Hence, when these solutions come together in PCR, the primers will anneal to the flanking sequence of the denatured DNA. Followed by extension by Taq DNA polymerase with the aid of dNTPs.

However, in the case of real-time PCR, probes are also added into the PCR master mix solution. These probes comprised of a reporter and quencher dye each at both ends. In addition, these probes sequences are complementary to the target DNA sequences. Therefore if the target DNA is present in the sample these probes will bind to it specifically and form a hybrid. However, the 5’ to 3’ nucleolytic activity of the Taq DNA polymerase will cause the hybrid (probe- target DNA) to cleave in between the reporter and quencher dye. A new extended strand of DNA will then displace the fragmented probes as the ploymerisation continues. Since the 3’ end of the probes is blocked, there will not be any amplifying of probe. The results is then read and recorded by software. The higher the fluorescence intensity indicated a higher amount of target DNA and vice-versa.

Designing the probes
Unlike other labs, my lab synthesizes our own probes. In order to synthesizes a probe there are certain guidelines to follow, such as
· GC contents of the probe must be within 20-80% range
· Identical nucleotide must be avoid
· Avoid a G nucleotide at the 5’ end
· Choose probes that have Cs instead of Gs
· The melting temperature should maintain at 68- 70 °C
· Probe sequence must be near to primer but not overlapping it

TaqMan Allelic Discrimination Kit
TaqMan Allelic Discrimination Kit is one of the most important kits I used during my research. It employed the principles used in the real-time PCR; only different is that it consists of two types of different probes instead of one. The two type of fluorescence probes used is TET (6-carboxy, 4,7,2,7’- tetrachlorofluorecein) while the other is FAM (6-carboxy fluorecein). These two probes is covalently linked to the 5’ end of the probes. Both type of probes will bind to their respective target DNA template (in this situation is two alleles) and form a hybrid. Fluorescence signals given by the reporter will only be detected when the DNA template is polymerized.

Take example; TET represents allele 1 and FAM represent allele 2. If a sample DNA template undergoes real-time PCR and high TET fluorescence signal is detected. This could mean that the individual is a homozygous for allele 1 and vice – versa for allele. However, what if both the signals are equally high?? Hmm…

Ans: That would indicate that the individual is a heterozygous (having both allele 1 and 2) (have you got it right?? Hehe)

Procedure for real-time PCR
1. Prepared the Real-time PCR master mix by adding the following:

· dNTPs,
· PCR buffer
· Probe
· RNase free water

2. Vortex the solution briefly
3. Add in the Taq DNA ploymerase
4. Inverted the centrifuge tube 2 to 3 times to mix the solution
5. Aliquot 9ml(for my lab) into each well in the 96 wells reaction plate/ MicroAmp optical tube
6. Close the plate tightly with the MicroAmp optical caps
7. Briefly centrifuge the plate (this is to allow the droplets cling on the side to settle)
8. Place the plate onto the thermal cycler block
9. Ensure that the real-time PCR cycling is in this manner:

· 25 ºC for 10 mins
· 48 º C for 30 mins
· 95 º C for 5 mins
10. Remove the plate and stored in fridge

Tips for real-time PCR
Here are some tips to reduce the chances of getting a false positive result. High DNA concentration from sample, amplicon carryover from previous PCR or positive control can lead to false positive results. In such case, AmpErase UNG (uracil N-glycoslase) treatment can be applied. This is because when UNG (uracil N-glycoslase) solution is added, the dTTPs are substitute by dUTPs in the PCR process. Hence it prevents the reamplification of the carryover in subsequent process by degrading the misprimer or non-specific PCR products via enzymatic reaction.

Another useful tip for PCR process is the addition of Q solution. Q solution changes the melting temperature of the DNA template during the PCR process. Therefore increase the specificity of the primers anneal to the DNA template during the PCR process. Besides that, it also increases the chances of achieving successful amplification and more PCR products.

By the way, if you have any question feel free to give me your comments.
Till then enjoy your SIP and see you all in the upcoming week!!

Avery, May lee(0503292E)
TG02

WEEK 3 Attachment Sharing

2007-07-12T01:56:00.000-07:00

Hello guys, it's my turn to share my SIP experience with you! =)

Anyway, for your information, i am currently working at a Oncology (cancer) research institute and everyone in my group is actually pretty much working on their own projects though we have general lab meetings every alternate weeks to update each other on our research progress etc etc.

As for me, i am focusing on a particular gene (let's just call it gene A) to see if there is an over-expression of it in prostate cancer. If there is a huge amount of gene A found in the prostate cancer cells, we can then make assumptions that perhaps this gene A has something to do with encouraging tumour angiogenesis or cancer cell proliferation etc etc. From it, we will then be able to find out if we could come up with an inhibitor that can block the action of the proteins (translated from gene A) and hopefully treat this cancer! (ok, this is the simplified version of my job scope. it's so much more complicated if i really want to elaborate but i will just leave it as that ok? =] )

For the past 3 weeks, i have been trained on Immunohistochemistry (IHC) which is very commonly used in cancer research to allow one to check if a particular protein is present/over-expressed in the cancer cells of that particular organ of the body. If you can still recall what we learnt during HistTech, it's 90% exactly the same as the staining we did =)

IHC: Materials and Methodology

1) As tissue samples from hospitals normally are formalin-fixed/paraffin-embedded sections, deparaffinization was done in 3 consecuetive xylene baths for 5 mins each

2) The slide was then dipped into 2 changes of absolute alcohol for 5 mins each as well.

Significance: To remove xylene from the slides and to prepare the sections for rehydration. This is because xylene and water are NOT miscible.

3) Rehydration was then performed as the slide was dipped in 95% alcohol (2 changes, 5mins each) before it was placed in 75% alcohol (2 changes, 5mins each). Rehydration is considered complete when the slide was rinsed with tapwater.

4) Pre-treatment was carried out as the slide was immersed in Ag Retrieval Buffer before it was placed in the Mega Microwave Histoprocessor at 117 degree Celsius for 30 mins.

Significance: When you fix a tissue sample using formalin, what it does is to denature proteins by coagulation, by forming additive compounds or by a combination of the 2. As a result, confomational changes in the protein structures will then occur to inactivate the enzymes and preserve the cells/tissues. Therefore, this pre-treatment process is to "revive" the immunoreactivity of the Ag to an immunogen. This process is aka "epitope retrieval" process.

5) The slide was cooled for 20mins before it was dripped dry and encircled using a Dako Pen

Significance: Dako pen is able to "trap" whatever reagents used on the sample within the encircled area so we don't have to use too much reagents and waste them.

6) Peroxidase block was added for 10 mins to the section to block any endogenous peroxidase that was produced by the cells.

Significance: Our secondary Ab will be labelled with Horseradish Peroxidase. Hence, we don't want any false positive results because of the presence of endogenous peroxidase.

7) Slide was washed to remove any excess peroxidase block reagent.

8) Ready-to-use Protein Block was used on the slide for 1 hour

Significance: Remember we have learnt about the "blocking" step in MBio last semester? It works in the same theory: to prevent non-specific binding.

DO NOT WASH SLIDE THIS TIME ROUND because even if you feel that the protein block might block the antigens in which the primary Ab have to bind to, this is not true. According to my senior, as the primary Ab used is more specific to the Ag than the protein block, it will have a higher affinity and the former can eventually replace the protein block. Hence, the end results will not be affected.

9) Primary Ab was then added to the slide for 1 hour. For every different kind of Ab used, we have a specific dilution for it. In case you are curious to know, i am using 1:5000 dilution =)

10) Washing was done this time to remove excess Ab

11) Secondary Ab labelled with HRP was added. Since my primary Ab is raised in mouse, my secondary Ab is an anti-mouse solution yup!

12) Washing carried out again

13) Chromogen and DAB was then added for just 5 minutes.

Significance: DAB will oxidise when it comes into contact with the air in the environment causing polymerisation and increasing its staining intensity. Chromogen is the substrate that will bind to the HRP to give us the signal we want.

14) After the slide was rinsed with tap water, it was counterstained using only Mayer's Haematoxylin.

Significance: As recent research has shown that protein A (from gene A) will be localised only in the nucleus, we only have to use haematoxylin and can save on our Eosin dye.

15) Dehydration was then carried out as the slide was dipped into 75% alcohol (2 changes, 5 mins each), 95% alcohol (2 changes, 5 mins each) and lastly absolute alcohol (2 changes, 5 mins each)

16) The slide was placed in the fume hood for 10 mins for drying to completely remove any water present on the slide/sample

Significance: water and xylene = NOT miscible

17) Lastly, the slide was placed in xylene for 3 changes for 5 mins each before it's mounted using DPX

Example:

Figure 1. Prostate cancer tumour sample expressing gene Z (Nucleus gives dark brown coloration). Note that only Mayer's Haematoxylin was used because researchers already know that gene Z staining is mainly localised in the nucleus.

Image is taken with permission from fellow co-worker.

Hope i didnt get my concepts wrong. LOL. correct me if you think i am inaccurate in any way yup =) Anyway, hope you guys are enjoying SIP over at your side.

Psst: should u have too much time to spare, u can join me for lunch coz i m alone like nisha as well! haha.

KangTing TG02

0503331A

Week 2 Attachment Sharing

2007-07-06T22:38:00.001-07:00

Hey there, i miss everyone because I'm alone here..haha, here's my part, if you don't understand, just ask me, if I never reply means I don't know what's the answer. lol

About my attachment

I’m attached to a research institute, in which all levels have research labs and different focus areas. I will be working on a paticular level throughout my attachment, and this level focuses on delivery of drugs, genes and proteins. Each student in my lab who are mainly university students and they are attached to a mentor, including myself and we are assigned onto a specific delivery. The major problem in current researches done throughout the world on such deliveries is that, the delivery efficiencies are low. Thus, we are trying to enhance the delivery of drugs and genes into cells for future cancer therapy and treatment of infectious diseases. Majority of the publications done by IBN are on cancer therapy.

Reseach Focus

My delivery focus is genes. These genes can be plasmid-encoding ones or siRNA. I’m working on how efficient the tranfected cells express the genes delivered to them and also the percentage of viability. Finally a comparison is done using different cell lines and nanocarriers. Let me explain the principle first.

Principle of the Project

This lab synthesizes nano-sized cationic polymers as carriers for the genes. The polymers have nitrogen group( NH₃⁺) which are positively charged that bind to the negatively charged phosphate group(PO₄^-) in the DNA. They form a transfection complex which will then be used to tranfect the cells. The uptake occurs via endocytosis. I will prepare the polymers and DNA solutions based on very confusing calculations in which I took almost a week to understand. The tranfection complexes are prepared in different conditions and pH(5&7). Conditions basically mean different nitrogen to phosphate ratio(N/P ratio: 0-40). I usually do 9-10 conditions. These are mainly done to determine the optimal concentration of the transfection complex and at what conditions do cytotoxicity occurs.

Lab techniques

Since I’m dealing with cells, I need to master all the cell culturing techniques and maintain my own cells. There are quite a no. of machine that I must learn to operate. Apart from that, I will have to prepare all my reagents by myself (labeled with my name) throughout my experiments and strictly no sharing.

Culturing and seeding of cells

After preparing all reagents and subculturing, I will seed the cells on 24 well plates or 96-well plates depending on which cell lines. Some cell line that I handle are HepG2, HEK 293, heLa and MCF-7 & etc. On the next day, I will do gene transfection on 1 cell line. The cell must meet the desired density, thus cell counting must be done using haemocytometer. Each condition has 4-6 replicates, thus every row of plate is for 1 condition. Then I will repeat the whole thing for another pH. The cells are then incubated in the CO₂incubator for 4 hrs and then the medium are changed to stop transfection. I will then need to wait for 3 days to do analysis using MTT assay and luciferase analysis assay.

Zeta potential and particle size

During the 3-day incubation, I will measure the zeta potential and particle size of the transfection complexes. Zeta potential basically means the surface charge of the particle. Since the cell surface are slightly –vely charged due to proteogylcans in the cell membrane, the complex must be more positive to enter the cell. However it must not be too high, because if too much complex enter the cells, cytotoxicity can occur. Apart from that, the particle size is also measured. It cannot be too large as they need to enter through the nuclear pores which are only 10nm in diameter. Both can be measured using a nanozeta and size analyzer based on different conditions and pH.

Assays for transfection efficiency and cytotoxicity & Total protein measurement

After 3 days, I can then do my analysis. I have not learnt the MTT assay procedures which is done to measure the cell viability, so I will explain about the luciferase analysis assay which can consume the whole day(seriously). This is done to determine the transfection efficiency(how many cells take up the complex). The DNA that I used for the complex is a luciferase-encoding plasmid. Cells that have been transfected will have the luciferase gene and can express the luciferase protein. The cells are then washed and lysed using lysis solution and further scrapped using pipette tips. This must be done properly as it will greatly affect the results. I took 3.5 hrs to do 4 24-well plates.

Then, I will measure the light intensity by adding substrate to the luciferase protein in the sample using the luciferase analyzer machine. A BSA standard is also done to convert the light intensity value to protein concentration. Apart from that, total protein content is also done to determine what is the percentage of luciferase protein over the total protein expressed by the cells. Both the BSA standard and total protein content are measured using 96-well plate reader.

From the beginning to the end of the experiment, I do have lots of excel sheets and graphs to plot. I should say that the steps and procedures are quite tedious and time consuming. Yet, IBN is good place for students like us because they give you the freedom to conduct any experiments you want, so long as it is within the focus area of the lab.

As you can see I did not explain about the step-by-step procedures for each phase because it is very long, so if you want to know, just send me a comment. I will blog more about result analysis and siRNA on my next post.

A brief one on LMQA

A*STAR divisions especially IBN put great emphasis on safety in the labs. Almost everyday, I do receive emails on safety aspects and every month, I have to attend safety briefing. I have an RFID card that I must always carry with me, so that if there is any emergency occurence, they can track where I am electronically. Apart from that, each lab has a SOP and incident occurrence box at the entrance. This white box contains the necessary documents to operate the machines, and also filing of accidents that have happened in the labs. Every 2 weeks, a safety officer will regularly check the boxes if all documents are present. Plus, at every level there is a MSDS room where you can find all the A-Z details of the chemicals. We are also given a small card containing a list of safety contact officers in case if there is any accident occurrence.

Regards,
Nisha Bte Mohd Rafiq
0503254E
TG02

Student Internship Programme Sharing

2007-07-01T04:16:00.000-07:00

Hi everyone, how's your first week of sip? Do share with me, ok?

I was being attached Histopathology/Cytology with June, Wing Fat and Desmond. For whole of 20weeks, we are being sent to the different stations of the histopathology laboratoy. For the first month, i am stationed at special staining, after which is cytology then processing next it will be embedding and microtomy and lastly we will take shifts to help in all the stations of the lab.

For special staining, i have learnt how to use and care for H&E (Haematoxylin&Eosin) machine and Ventana special stainer machine. It also includes batch collection and mangement of slides. Im also expected to pick up special histological staining techniques to produce good quality stained sections.

I also read up quality management system, environmental management(ISO 1400I requirements), emergency actions, safe disposal of waste, sources of contamination and suggestions to prevent it from the medical technologists in the lab. Some of these can be found in our LMQA notes. We do have to follow good lab management in order to have quality assurance.

Histopathology Lab Process
1) Customer services (hospitals)
2) Histology lab reception (specimen identification and sorting by medical technologists)
3) Accessioning (numbering and sorting request forms)
4) Tissue Processing
5) Microtomy
6) Special staining/routine H&E stains (Haematoxylin & Eosin)
7) Mounting
8) Sorting and labeling of stained stains
9) Dispatch slides
10) Reporting by pathologist
11) Typing
12) Verification
13) Electronic signed-out
14) Reports dispatched-out

Hot plate
- Up to 30 tissue samples slides can be placed on the hot plate
- Sample slides are placed on the hot plate for at least 3minutes
- Small tissue sample slides must be totally dried before placing onto hot plate so to prevent floating

Routine Auto-Stainer
For routine Haematoxylin & Eosin steps involves:
1. Xylene
2. Xylene
3. Absolute alcohol
4. 95% alcohol
5. 70% alcohol
6. Water
7. Haematoxylin
8. Haematoxylin
9. Water

Ventana automated special stainer
- An automated system that is barcode-driven with optimized protocols
- Helps to increase productivity and efficiency
- Image link: http://www.ventanamed.com/ (NEXES special stain)
- Special stain wash solution is required to spray onto the slides to hydrate the tissue samples to prevent them from drying up as it may affect staining
- Example of special stains carried out: PAS(Periodic Acid Schiff), PASD, GMS(Gomori’s Methenanine Silver Nitrate) for fungus, Re (Retic), Fe (iron), Giemsa, Steiner, Alcian Blue
- Slide tray holds up to 20 test slides
- Reagent tray holds up to 25 reagent
1) Key in barcode labels and protocol(test selected) for the slides that are being used
2) Put slides in slide tray
3) Load reagents required
4) Put back reagent tray and open up all reagents’ cover
5) After which, off preheat as preheat is on before machine is used
6) Then click Run
7) Make sure all criteria of pre-run checklist is met
8) Key in total number of slides being processed
9) Run time is shown
10)When the staining process is done, remove reagents and slides
11)Click sign off
12)Slides ready

This is one of the special stains that is being done manually: Test for tuberculosis (TB)- Ziehl Neelsen
TB test/ Zn : stains for tubercle bacilli head : red
Steps
1) Sections to water
2) Commercial TB colour (5mins)
3) Wash in water (running water)
4) Differentiate with 1% acid alcohol (until colorless/light pink)
5) Wash in water
6) Counterstain with 1% Loeffler’s methylene blue (5-10sec) until sky blue
7) Wash in water and go straight to 95% alcohol to control blue color (if very blue, use 70% alcohol)
8) Dehydrate, clear and mount

Sorting of slides
- 10% of the 30 slides are checked for completeness and if they are satisfactory; if they are unsatisfactory or did not meet the guidelines, they will be sent for re-cut (corrective action)
- Is part of quality check
- This is to safeguard the patients’ interest
- Check ready slides against tissue blocks to ensure that both are from the same source
- Request forms that are sent out with ready slides are to be stamped by Amano Pix 3000 with date and time

This is what i have went through in my first week of SIP and this shall be the end of my first post. I shall update more the next time.

Ang Xiao Si Sharon
0503219H
TG02

~shortlisted disease~ --- Explanation

2007-05-13T18:58:00.001-07:00

1) Murine Typhus

Causative agent : Rickettsia typhi present in the infected flea can be directly transmitted into the patient’s skin .

Reasons: In this case, the patient has been feeding stray cats and dogs; there is a high probability that the animals have been the vehicles to transmitted ticks on to the man. Hence, an infected tick could have bitten the man.

Sample(s) to be taken: Blood

Collection: Via venipuncture

Preliminary testing:Giemsa staining for light microscopy

2) Lyme disease

Causative agent : Lyme disease is caused by the bacterium Borrelia burgdorferi.

Reasons: Since the patient had the habit of feeding stray animals, it might have provided an opportunity for the bacterium Borrelia burgdorferi to transmit from the animals to him.

Sample(s) to be taken: Blood

Collection: Via venipuncture

Preliminary testing:Physical examination for symptoms and ELISA

3) Typhoid fever

Causative agent :Typhoid is a disease caused by Salmonella typhi.

Reasons: Since the patient living condition was so poorly sanitary. There is a high chance that the stray animals and cockroaches come into contact with the sewage and contaminated the food.As a result lead to faecal-oral route contamination of food.

Sample(s) to be taken: Blood or feces.

Collection: Via venipuncture and collection containers

Preliminary testing: Gram-stained smear or TSI agar

~shortlisted disease~

2007-05-01T20:38:00.000-07:00

1) Murine Typhus

2) Lyme disease

3) Typhoid fever

MMic tut 1-4: A stimulated Case

2007-04-24T19:59:00.000-07:00

List of possible diseases

a)Nisha

1)Viral hemorrhagic fever is caused by ebola virus. Often found in rodents.

Link : http://healthlink.mcw.edu >article>955159073.html

2)Typhus is caused by a bacteria that can be found in rats and Can be transmitted to human due to poor personal hygiene. Symptoms are fever and rashes.

Link : http://en.wikipedia.org >wiki>Epidemic_Typhus

3)Leprosy is caused by a mycobacteria that can infect people living in poor conditions and sanitization( higher risk). Symptoms are rashes and body weakness.

Link : http://en.wikipedia.org >wiki>Leprosy

b)Nur Zahirah

1) Plague is caused by a bacterium Yersinia pestis and people get infected when they got bitten by the infected flea or when handling animal. Symptoms include fever

Link : http://www.cdc.gov >ncidod>dvbid>plague

2)Leptospirosis caused by bacteria of the genus Leptospira. Dogs in urban or rural areas can carry the bacteriumSymptoms include high fever and rash.

Link : http://www.orlandorats.com >diseases.htm

3)Typhoid fever caused by a bacteria called Salmonella typhi that is found in the stools of infected person. Symptoms include fever, skin rash and weakness

Link : http://www.metrokc.gov >health>prevcont>typhoid.htm#diagnosis

c) Sharon

1)Bubonic plague is the best-known variant of the deadly infectious disease plague, which is caused by the enterobacteria Yersinia pestis. The epidemiological use of the term plague is currently applied to bacterial infections that cause buboes, although historically the medical use of the term plague has been applied to pandemic infections generally.

Link : http://en.wikipedia.org >wiki>Bubonic_plague

2) Typhus fever is a potentially fatal, infectious disease caused by the bacterium Rickettsia prowazekii. Also known as epidemic typhus, this disease is transmitted to humans by body lice. Though it has occurred on all continents throughout the world except Australia, typhus fever is not common in the United States.

Link : http://www.azdhs.gov >phs>edc>edrp>es>typhusfeverf.htm

d) KangTing

1) Murine typhus is an infectious disease that is spread by lice or fleas. Some symptoms for this disease include extremely high fever (105 to 106 degrees Fahrenheit), which may last up to 2 weeks and dull red rash that begins on middle of the body.

Link : http://www.nlm.nih.gov >medlineplus>ency>article>001363.htm

2) Plague is an infectious disease of animals and humans caused by a bacterium named Yersinia pestis. It is a flea-borne disease and can be transmitted via respiratory droplets from cats and humans with pneumonic plague.

Link : http://www.cdc.gov >ncidod>dvbid>plague>facts.htm

3) Lyme disease is caused by the bacterium Borrelia burgdorferi and is transmitted to humans by the bite of infected blacklegged ticks. Some og the symptoms include a circular rash called erythema migrans or EM. Patients also experience symptoms of fatigue, chills, fever, headache, and muscle and joint aches, and swollen lymph nodes.

Link: http://www.cdc.gov>ncidod>dvbid>lyme>ld_humandisease_symptoms.htm

e) May Lee

1)Hookworm infection is a disease caused by infestation with the roundworms Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, or Ancylostoma braziliense. The symptoms of animal hookworm infection in people can get painful and itchy skin infections. Mainly caused by the eggs can hatch into larvae, and both eggs and larvae which can be found in dirt where animals have been.

Link : http://www.nlm.nih.gov > medlineplus>ency>article>000629.htm
Link : http://en.wikipedia.org >wiki>Hookworm_infections

2) Colorado tick fever is an viral infection transmitted by the bite of the Dermacentor andersoni tick. The symptoms includes headache, fever, rash and muscle pain.

Link : http://www.nlm.nih.gov > medlineplus>ency>article>000675.htm
Link : http://en.wikipedia.org >wiki> Colorado_tick_fever

3) Trichinosis is an infection cuased by the roundworm Trichinella spiralis it is usually infecting by consuming undercooked meat containing this microbe. High fever ,muscle pain and Pink eye (conjunctivitis) are some of the common symptoms experiences when infected with Trichinosis.

Link : http://www.nlm.nih.gov > medlineplus>ency>article> 000631.htm

f) Johanna

1)Causative agent: Bite from Ixodes Tick transmitting the bacterium Borrelia burgdorferi
Disease caused: Lyme disease
Known symptoms: Fever, headache, stiff neck or neck pain, fatigue, erythema migrans
(slowly-expanding “ bull’s-eye” rash)
(observed symptoms are in bold)

Reasons for diagnosis: Lyme disease is normally diagnosed by symptoms, physical findings (eg. rash) and the possible exposure to infected ticks. Since the 85 year-old man has been feeding stray cats and dogs, there is a high probability that the man could have been bitten by an infected tick.

Physical examination: Main method of diagnosis. At the early stage of the disease, serological testing is usually not done. Furthermore, patients of EM rash are not ususally tested on to determine the diagnosis. In both the early and late stage of the disease, there is a high possibility of false negatives. Late stage symptoms will be more difficult to diagnose due to the variety of symptoms that overlap with other better known diseases.

Serological tests: Western Blotting, Enzyme-linked Immunosorbent Assay (ELISA), Polymerase Chain Reaction (PCR) etc.

Link : http://en.wikipedia.org > wiki>Lyme_disease
Link : http://www.cdc.gov >ncidod>dvbid>lyme>

Our Group Expectations

2007-04-24T19:34:00.000-07:00

With 6 people being posted to different locations for our Student Internship Programme, we hope this blog will not only allow us to keep in touch with each other for the 20 weeks but also allow us to learn from each other's experiences.

We will try to post interesting blog entries as frequent as possible so that not only will our group members benefit from all the information posted but anyone who reads our blog will enjoy reading them! =D

Always remember: LEARNING IS FOR LIFE!

Microorganism	Test	Result
Staphylococcus aureus	● Gram-staining ● Culturing on mannitol salt/blood agar ● Coagulase test ● Catalase test ● TSI	● Gram-positive, cluster-forming cocci ● Yellow or gold colonies, drop in pH (yellow area) / ß-hemolytic ● Positive ● Positive ● Acid slant/acid butt
Enterococci	● Gram-staining ● Catalase test	● Gram-positive cocci, occuring singly, in pairs, or in short chains ● Negatives
Coagulase-negative staphylococci	● Gram-staining ● Culturing on blood agar ● Coagulase test ● Catalase test	● Gram-positive, cluster-forming coccus ● Yellow or gold colonies ● Negative ● Positive
Escherichia coli	● Gram-staining ● Culture on EMB/ MacConkey's agar ● TSI agar ● Urease test ● Indole test ● Citrate test	● Gram-negative rod ● EMB:Lactose-fermenting, blue-black colonies with metallic green sheen ● MacConKey:Lactose-fermenting, red colonies ● Acid slant/acid butt with gas but no H2S ● Negative● Positive ● Negative
Pseudomonas aeruginosa	● Gram-staining● Culture on EMB/ MacConkey's agar ● TSI● Oxidase test ● Indole test ● Citrate	● Gram-negative rod ● EMB/MacConkey:Non-lactose fermenting colonies ● Alkaline slant/alkaline butt ● Positive ● Negative ● Positive
Enterobacter species	● Gram-staining ● Culture on EMB/ MacConkey's agar ● Urease test ● Vogues-Proskauer test ● Citrate test	● Gram-negative rod ● EMB: Lactose-fermenting, brown dark -centered, mucoid colonies ● MacConkey:Lactose-fermenting, pink mucoid colonies ● Negative ● Positive ● Positive
Proteus mirabilis	● Gram-staining ● Culture on EMB/ MacConkey's agar ● TSI ● IMVIC ● Urease test	● Gram-negative cocci ● EMB/MacConkey:Non-lactose fermenting colonies ● Alkaline slant/acid butt with H2S ● Indole: Negative ● Methyl-red: Positive ● Vogues-Proskauer: Negative ● Catalase: Positive ● Positive
Klebsiella pneumoniae	● Gram-staining ● Culture on EMB/ MacConkey's agar ● TSI ● Indole test ● Urease test ● Citrate test	● Gram-negative rod ● EMB: Lactose-fermenting, brown dark -centered, mucoid colonies ● MacConkey:Lactose-fermenting, pink mucoid colonies ● Acid slant/acid butt with some gas production, no H2S ● Negative ● Positive ● Positive

Gram-negative	Test	Result
Gardnerella vaginalis	● Morphology ● Oxidase ● TSI ● IMViC ● Laboratory diagnosis /culture	●Bacilli ● negative ● Acidic slant/acidic deep ● Catalase (-) ● Chocolate agar and HBT agar: Small, circular, convex, gray colonies ●Colistin-oxolinic acid blood agar: Beta-hemolysis
Escherichia coli	● Morphology ● Oxidase ● TSI ● IMViC ● Laboratory diagnosis /culture	●Bacilli ● negative ● Acidic slant/acidic deep ● I(+),M(+), Vi(-),C(-), U(-) ● EMB: green sheen,fermenting colonies ●MacConkey agar: fermenting colonies
Neisseria gonorrhoeae	● Morphology ● Oxidase ● TSI ● Laboratory diagnosis /culture	●Bacilli ● positive ● Acidic slant/acidic deep ●Giemsa-stained ●PCR and ELISA ●Immunofluorescence
Chlamydia trachomatis	● Morphology ● Oxidase ● Laboratory diagnosis /culture	●cocci ● positive ●Giemsa-stained ●PCR and ELISA ●Immunofluorescence
Pseudomonas aeruginosa	● Morphology ● Oxidase ● TSI ● IMViC ● Laboratory diagnosis /culture	●Bacilli ● positive ● Alkaline slant/ alkaline butt ●Catalase (+) ●EMB and MacConkey agar: non-fermenting colonies

Gram - positive	Enterococcus faecalis	Staphylococcus saprophyticus
Morphology	Cocci( in pairs)	Bacilli
Catalase test	-	+
coagulase	Nil	-
Laboratory diagnosis /culture	Blood agar: non-hemolysis	Mac Conkey’s agar: Spherical, irregular grape-like cluster in culture

Other microbes	Trichomonas vaginalis	Candida albicans	Mycoplasma hominis
Morphology	acridine orange : pear-shaped, motile, flagellated protozoan	single-celled, diploid fungus	round, pear shaped and even filamentous
Laboratory diagnosis /culture	Trichomonas Direct Enzyme Immunoassay and Fluorescent Direct Immunoassaysaline wet preparation : motile trichomonads and increased PMNs (ratio of PMNs to vaginal epithelial cells)	Blood agar plates: large, round, white or cream colonies	Mycoplasma GU Culture System: ‘fried egg’ and granular appearance colonies