J.A.M.M.Y.S

MMIC PBL Package 2

2008-01-24T06:05:00.000-08:00

Pathogens that cause infections during jungle training

Viruses

Organism:
Hepatitis A virus (picornavirus)
Mode of transmission:
Fecal-oral route, direct contact with an HAV-infected person, ingestion of contaminated food or water prepared by a HAV-infected person
Disease(s) caused:
Hepatitis A
Signs & symptoms:
Jaundice, fever, anorexia, nausea, vomiting, diarrhea, myalgia, malaise, dark-coloured urine or light-coloured feces, abdominal tenderness, hepatomegaly or spleomegaly
Prevention:
Hepatitis A vaccination, short-term protection from immunoglobulin (can be given before and within 2 weeks of coming in contact with HAV), good personal hygiene and proper sanitation

Organism:
Hepatitis B virus
Mode of transmission:
Blood contact from a HBV-infected individual, unsafe injection
Disease(s) caused:
Hepatitis B
Signs & symptoms:
Yellowing of skin and eyes (jaundice), extreme fatigue, abdominal pain, loss of appetite, nausea, vomiting, joint pain
Prevention:
Hepatitis B vaccination, avoid sharing needles, syringes with HBV-infected indivduals, avoid sharing personal care items that might have blood on it, such as razors, toothbrushes

Organism:
Hepatitis E virus
Mode of transmission:
Ingestion of contaminated food or water, contact with HEV-contaminated blood
Disease(s) caused:
Hepatitis E
Signs & symptoms:
Jaundice, fatigue, abdominal pain, loss of appetite, nausea, vomiting, dark urine (tea coloured)
Prevention:
Avoid drinking water of unknown purity, uncooked shellfish, and uncooked fruits or vegetables that are not peeled or prepared hygienically

Organism:
Rabies (Mononegavirales species)
Mode of transmission:
Via bites and virus-containing saliva of an infected host (dogs, cats, foxes, insectivorous and vampire bats etc.), contaminated mucous membrane such as eyes, nose, mouth, aerosols
Disease(s) caused:
Rabies
Signs & symptoms:
Non-specific flu-like symptoms: malaise, fever, headache, discomfort at site of infection, anxiety, hallucination, insomnia
Prevention:
Anti-rabies vaccination, wash wound thoroughly with soap and water, detergent or plain water, followed by the application of ethanol, tincture or aqueous solution of iodine

Organism:
Influenza virus (type A strain)
Mode of transmission:
Farm-to-farm by the movement of live-infected birds, movement of people (especially with contaminated shoes and clothes), contact with contaminated vehicles, equipments and cages, contact with bird feces, swimming in rivers where carcasses of dead infected birds are discarded
Disease(s) caused:
Avian flu
Signs & symptoms:
Initial symptoms: high fever, usually with a temperature higher than 38˚C and influenza-like symptoms, watery diarrhoea, vomiting, abdominal pain, chest pain and bleeding from the nose and gums can be seen during the early stages
Later stage: lower respiratory tract problem: difficulty in breathing, respiratory distress, a hoarse voice and a crackling sound when inhaling, sputum production is variable and sometimes bloody
Prevention:
Avoid direct or close contact with infected poultry or surfaces contaminated with secretion and excretion from infected birds, wash hands with soap and warm water for at least 20 seconds before and after handlilng raw poultry and eggs, ensure poultry food products are well-cooked

Organism:
Nipah virus
Mode of transmission:
Close contact with contaminated tissue or body fluids from infected animals (eg. pigs)
Disease(s) caused:
Nipah virus disease and encephalitis
Signs & symptoms:
Mild or asymptomatic: high fever and muscle pains, prolong infection may result in inflammation of the brain (encephalitis) with drowsiness, disorientation, convulsion and coma
Prevention:
Avoid close contact with infected animals, always have good hygiene practice

Organism:
Chikungunya virus (CHIKV) (genus – Alphavirus, family - Togaviridae)
Mode of transmission:
The bite of an infected Aedes mosquito (most commonly Aedes aegypti, less commonly Aedes albopictus
Disease(s) caused:
Chikungunya fever
Signs & symptoms:
Fever (can reach up to 39°C), headache, fatigue, nausea, vomiting, muscle pain, rash, and joint pain insomnia. However Silent CHIKV infections (infections without illness) do occur.
Prevention:
No vaccine for chikungunya fever is available. Precautionary measures includes the prevention of mosquito bites by wearing long sleeves and pants and/or using an EPA-registered insect repellent such as those with DEET, picaridin or oil of lemon eucalyptus on exposed skin.

Organism:
Dengue virus (consists of four closely related virus serotypes [DEN-1, DEN-2, DEN-3, and DEN-4] of the genus Flavivirus)
Mode of transmission:
The bite of an infected Aedes mosquito (most commonly Aedes aegypti, less commonly Aedes albopictus])
Disease(s) caused:
Dengue fever (DF) or dengue hemorrhagic fever (DHF)
Signs & symptoms:
Fever (ranging from a mild to an incapacitating high fever), severe headache, pain behind the eyes, rashes and muscle pain and joint pain. DHF (fever, abdominal pain, vomiting, bleeding) is a potentially lethal complication.
Prevention:
No specific antiviral medicines or vaccination for dengue is available due to the many virus serotypes present. Precautionary measures includes the prevention of mosquito bites by wearing long sleeves and pants and/or using an EPA-registered insect repellent such as those with DEET, picaridin or oil of lemon eucalyptus on exposed skin.

Organism:
Japanese encephalitis (JE) virus (a Flavivirus antigenically related to St. Louis encephalitis virus
Mode of transmission:
The bite of infected rice field breeding mosquitoes (primarily the Culex tritaeniorhynchus group)
Disease(s) caused:
Japanese encephalitis
Signs & symptoms:
Acute encephalitis (inflammation of the brain), fever, headache malaise neck rigidity, cachexia (loss of weight, muscle atrophy, fatigue, weakness and significant loss of appetite), hemiparesis (partial paralysis of one side of the body), convulsions and a raised body temperature between 38 and 41 degrees Celsius can progress to paralysis, seizures, coma and death.
Prevention:
An inactivated Japanese encephalitis(JE) vaccine is available. The vaccine is reactogenic, however the rates of serious allergic reactions (generalized urticaria or angioedema) are low. Precautionary measures includes the prevention of mosquito bites by wearing long sleeves and pants and/or using an EPA-registered insect repellent such as those with DEET, picaridin or oil of lemon eucalyptus on exposed skin.

Organism:
Yellow fever virus (a Flavivirus)
Mode of transmission:
The bite of an infected mosquito (Transmission from human to human is mostly mediated by the Aedes aegypti mosquito)
Disease(s) caused:
Yellow fever
Signs & symptoms:
Initial phase which includes symptoms of fever and chills, severe headache, back pain, general muscle aches, nausea, fatigue, and weakness can be followed by followed by a short period of symptom remission.
Afterwards the toxic phase develops as the fever returns, with clinical symptoms including high fever, headache, back pain, nausea, vomiting, abdominal pain, and fatigue.
Hepatic coagulopathy may also occur causing hemorrhagic symptoms, including hematemesis (black vomit), epistaxis (nose bleed), gum bleeding, and petechial and purpuric hemorrhages (bruising). Deepening jaundice (the symptom which led to the name of the condition) and proteinuria frequently occur in severe cases.
Prevention:
Yellow fever vaccine, which is a live virus vaccine, is available. The vaccine generally has few side effects causing mild headache, muscle pain, or other minor symptoms in fewer than 20% of patients. Persons allergic to eggs should not be vaccinated as the vaccine is prepared in eggs and immunosuppressed individuals should not be vaccinated due to the nature of the live virus vaccine. Precautionary measures includes the prevention of mosquito bites by wearing long sleeves and pants and/or using an EPA-registered insect repellent such as those with DEET, picaridin or oil of lemon eucalyptus on exposed skin.

Extra Precautions:

There is an increased risk of mosquito-borne diseases during and immediately after the rainy season thus take extra precaution during this period.
Infected individuals should be protected from further mosquito exposure (staying indoors and/or under a mosquito net during the first few days of illness) so that they can't contribute to the transmission cycle.

* Done by: Azhar & Yvonne

Fungi

Fungi are often present in the environment such as in the soil and in decaying vegetation with the exception of Candida albicans, which is part of normal human flora. As the army is training in the jungle where they are constantly exposed to the fungi present in the environment, there is a high risk of them getting fungal infections.

Organism:
Trichophyton mentagrophytes (dermatophytes)
Epidemiology:
World wide
Mode of transmission:
Direct contact with skin lesions of infected people, mice and rodents or with infected keratin debris on the ground
Disease(s):
Athlete’s foot (tinea pedis)
Signs & symptoms:
Itching and burning feet, skin frequently peels
Prevention:
Wear breathable shoes, wear cotton socks and change after sweating, ensure that shoes are dry before tying the shoe lace, always keep the feet dry and use talcum powder if required

Picture is taken from http://www.lib.uiowa.edu/hardin/md/cdc/4803.html

Organism:
Sporothrix schenckii
Epidemiology:
Temperate and tropical regions
Mode of transmission:
Spores present in rose thorns, sphagnum moss, soil and decaying vegetation enter the skin through exposed wounds
Disease(s):
Sporotrichosis
Signs & symptoms:
Small painless bump or lesion at the site of wound, the colour ranges from red to purple. The lesion will then grow larger and discoloured, eventually developing into an ulcer when left untreated
Prevention:
Wear gloves and long sleeves, avoid skin contact with sphagnum moss and soil especially when there is a wound

Picture is taken from http://www.healthinplainenglish.com/health/infectious_diseases/sporotrichosis/

Organism:
Cryptococcus neoformans
Epidemiology:
World wide
Mode of transmission:
Inhalation of airborne yeast cells, commonly found in soil with pigeon droppings. It can also be found in unwashed raw fruits.
Disease(s):
Cryptococcosis
Signs & symptoms:
Asymptomatic in healthy individuals
Prevention:
Immunocompromised individuals should avoid areas contaminated with pigeon or other bird droppings, such as barns and areas under bridges where pigeons roost

Picture is taken from http://www.medscape.com/viewarticle/410174

Organism:
Candida albicans
Epidemiology:
World wide
Mode of transmission:
Present as normal flora on skin, mucous membranes and GI tract
Disease(s):
Thrush, mucosal and systemic candidiasis
Signs & symptoms:
Depends on the type of disease
Prevention:
Reduce predisposing factors (eg. severe trauma, burns), oral thrush can be prevented by using clotrimazole troches or nyastin

Picture is taken from http://www.tonguethrush.com/
An example of tongue trush.

Organism:
Conidiobolus coronatus (zygomycetes)
Epidemiology:
World-wide but mainly in tropical areas such as Indonesia, Southeast Asia and Africa
Mode of transmission:
Contact with soil and decaying vegetation or introduction of spores into nasal mucosa that has a trauma (eg. caused by an insect bite)
Disease(s):
Subcutaneous zygomycosis (chronic inflammatory or granulomatous infection restricted to the nasal mucosa)
Signs & symptoms:
Fever, lethargy, headache

Organism:
Aspergillus fumigatus
Epidemiology:
Temperate regions
Mode of transmission:
Inhalation of airborne spores from soil and plant debris
Disease(s):
All forms of non-invasive and invasive aspergillosis
Signs & symptoms:
Non-invasive (fever, malaise, coughing out blood), invasive (chills, headache, increased production of sputum which may be bloody, chest pain)

Picture is taken from http://medimages.healthopedia.com/large/aspergillosis.jpg

Prevention:

- Wear clean, dry, loose-fitting clothes whenever possible

- Avoid sleeping in wet clothes

- Keep short hair

* Done by: Ming Boon & Michelle

Protozoa

Organism:
Plasmodium species (P. falciparum, P. vivax)
Epidemiology:
World-wide
Mode of transmission:
From infected female Anopheles mosquitoes to human
Disease(s):
Malaria
Signs & symptoms:
Fever, chills, intravascular hemolysis & cerebral malaria (P. falciparum infections)
Prevention:
Vector control using DDT, organochlorine and organophosphate mosquito control insecticides, anti-malarial drugs

Organism:
Entamoeba histolytica
Epidemiology:
World-wide with a higher prevalence in tropical countries
Mode of transmission:
Ingestion of cysts transmitted by fecal-oral route in contaminated food and water, person-to-person, animal-to-human (pigs & monkeys)
Disease(s):
Amebiasis
Signs & symptoms:
Dysentery (bloody, mucous containing stools, lower abdominal pain, tenesmus), liver abscess (right upper quadrant pain, weight loss, fever, tender and enlarged liver)
Prevention:
There are no effective immunizations and prophylaxis and thus only preventive measures are limited to personal hygiene

Organism:
Giardia species (G. lamblia & G. duodenalis)
Epidemiology:
World-wide
Mode of transmission:
Ingestion of cysts transmitted by fecal-oral route in contaminated food and water, person-to-person
Disease(s):
Giardiasis
Signs & symptoms:
Foul-smelling, loose and greasy stools
Prevention:
Drink boiled, filtered and iodine-treated water

Organism:
Toxoplasma gondii
Epidemiology:
World-wide
Mode of transmission:
Ingestion of undercooked meat, food and water contaminated with cat feces (fecal-oral route)
Disease(s):
Toxoplasmosis
Signs & symptoms:
Often asymptomatic, immunocompetent individuals may have lymphadenopathy, fever, headaches and muscle aches
Prevention:
Avoid contact with cat feces, cook food and boil water thoroughly

Organism:
Cryptosporidium parvum
Epidemiology:
World Wide
Mode of transmission:
Ingestion of contaminated food or water and exposure to feces (Water and food borne, zoonotic)
Disease(s):
Cryptosporidiosis
Signs & Symptoms:
Diarrhea, nausea, vomiting, fever, weight loss, dehydration
Prevention:
Good hygiene practices, drink boiled water, avoid exposed feces, uncooked food

Organism:
Cyclospora cayetanensis
Epidemiology:
World Wide
Mode of transmission:
Ingestion of contaminated food or water and exposure to feces (Water and food borne, zoonotic)
Disease(s):
Cyclosporiasis
Signs & Symptoms:
Diarrhea, stomach bloatness, nausea, loss of appetite, low grade fever
Prevention:
Good hygiene practices, drink boiled water, avoid exposed feces, uncooked food

Organism:
Balantidium coli
Epidemiology:
Tropics
Mode of transmission:
Ingestion of food or water contaminated by human or animal feces containing B. coli cysts. (Water and food borne, zoonotic)
Disease(s):
Balantidiasis
Symptoms:
Dysentery, abdominal pain, colitis
Prevention:
Purification of water, proper food handling, careful with feces disposal

Organisms:
Enterocytozoon bieneusi, Septata intestinalis
Epidemiology:
World wide
Mode of transmission:
Water and food borne, zoonotic
Disease(s):
Microsporidia
Signs & Symptoms:
Conjunctivitis, keratoconjunctivitis, bronchiolitis, pneumonia, rhinosinusitis, disseminated infection
Prevention:
Purification of water, proper food handling

Organism:
Trypanosoma cruzi
Epidemiology:
Latin America and tropical rainforest
Mode of transmission:
Insert borne (triatomine bugs) and food borne
Disease(s):
Chagas’ disease
Signs & Symptoms:
Acute: fever, fatigue, body aches, headache, rash, loss of appetite, diarrhea, and vomiting, mild enlargement of the liver or spleen, swollen glands, and local swelling
Prevention and Precaution:
Spraying infested dwellings, wearing protective clothing, and applying insect repellent to exposed skin and bed net

* Done by: Kau Hin, Ming Boon

Bacteria

Organism:
Leptospira interrogans
Epidermiology:
World wide especially in tropical areas and most particularly Southeast Asia
Mode of transmission:
Contact (mucous membrane exposure) or ingestion of urine of infected animals (rodents, wild mammals & domestic animals) contaminated food
Disease(s):
Leptospirosis
Signs & symptoms:
Biphasic, early (fever, chills, intense headache & conjunctival suffusion), second (aseptic meningitis, jaundice & renal failure)
Prevention:
Difficult because the organism is not eradicated from wild animals which constantly infect domestic animals. Use of doxycycline, practice good personal hygiene, avoid swimming in streams where risk of infection is high, avoid drinking of water from stream

Helminths

Organism:
Schistosoma japonicum (flatworm)
Mode of transmission:
Penetration through the skin when contact with infected snails
Disease(s):
Schistosomiasis, affecting the GI tract
Signs & symptoms:
Itching, dermatitis, fever, chills, diarrhea, lymphadenopathy and hepatosplenomegaly
Prevention:
Avoid swimming in affected areas, eliminating snail host when possible, proper disposal of human waste

Organism:
Wuchereria bancrofti, Brugia malayi
Mode of transmission:
Bite of female Anopheles and Aedes mosquito
Disease(s):
Filariasis
Signs & symptoms:
Elephantiasis (massive edema), coughing & wheezing at night, fever
Prevention:
Use of proper clothing, mosquito netting and repellent

* Done by: Shahirah

References:
1. http://www.healthinplainenglish.com>health>infectious diseases>sporotrichosis
2. http://www.treatnailfungus.org>treatment>athlete's foot fungus
3. http://www.emedicine.com>name of disease
4. http://mosqpro.com>mosquito borne diseases
5. http://www.mycology.adelaide.edu.au>mycoses 6. http://gsbs.utmb.edu>micro book>chapter 79 7. http://www.cdc.gov>virus name 8. http://www.who.int>dengue 9. Levinson W. 2006. Review of Medical Microbiology and Immunology, 9^th edition. McGraw Hill Companies

2007-12-09T17:16:00.000-08:00

Case 1 (Jiaxin)

Patient: Khong Fay Seah, Female, 27, outpatient

Complaints: Fever, chills, dysuria
Diagnosis: Urinary Tract Infection (UTI)

	Gram Stain	Oxidase	Lactose	TSI	Indole	Methyl-red	Voges-Proskauer	Citrate	Urease	Wet Mount	Antibiotic Susceptibility Tests
Escherichia Coli	-	-	+	slant & butt yellow (acidic), gas produced, H2S negative	+	+	-	-	-	Motile bacillus	Penicillin G (Ampicillin), Sulfonamides Cephalosporin, Aminoglycoside
Proteus mirabilis	-	-	-	slant red (alkaline) & butt yellow (acidic), gas & H2S produced	-	+	-	+	+	Highly motile bacillus	Trimethoprim-sulfamethoxazole, Ampicillin
Klebsiella pneumoniae	-	-	+	slant & butt yellow (acidic), gas produced, H2S negative	-	-	+	+	+	Non-motile bacillus	Penicillin, Cephalosporin, Aminoglycoside, Cephalosporin (e.g. Cefotaxime)
Enterobacter spp	-	-	+	slant & butt yellow (acidic), gas produced, H2S negative	-	-	+	+	-	Motile bacillus	Ciprofloxacin, Aminoglycoside (e.g. Gentamycin)
Pseudomonas aeriginosa	-	+	-	slant & butt red(alkaline), gas & H2S negative	+	-	-	+	-	Motile bacillus	Penicillin, Cephalosporin, Aminoglycoside, Tetramycin

Table1.1 Bichemical, Microscope and Antibiotic Susceptibility Tests Result for Gram-negative Mibcrobes

	Gram Stain	Catalase	Coagulase	Hemolytic on Blood Agar	Wet Mount	Antibiotic Susceptibility Tests
Staphylococcus saprophytcus	+	+	-	Non-hemolytic	Non-motile cocci	Norfloxacin, Trimethoprim-sulfamethoxazole (e.g. Bactrim)
Staphylococcus aureus	+	-	+	Beta hemolytic colonies	Non-motile cocci	Vancomycin, Amoxicillin with clavulanic acid, Cloxacillin, Ampicillin, Cephalosporon
Enterococcus faecalis	+	-	N.A	Non-hemolytic colonies (24 hours) Alpha-hemolytic(48 hours)	Non-motile cocci	Ampicillin, Vancomycin, Aminoglycoside

Table1.2 Bichemical, Microscope and Antibiotic Susceptibility Tests Result for Gram-positive Mibcrobes

Cause the previous post's table was cut off, this is the better version. Thanks

MMIC blog post 2

2007-12-09T04:15:00.000-08:00

Case 1

Patient: Khong Fay Seah, Female, 27, outpatient

Complaints: Fever, chills, dysuria

Diagnosis: Urinary Tract Infection (UTI)

Gram Stain	Oxidase	Lactose	TSI	Indole	Methyl-red	Voges-Proskauer	Citrate	Urease	Wet Mount	Anitbiotic Susceptibility Test
Escherichia Coli	-	-	+	slant & butt yellow (acidic), gas produced, H₂S negative	+	+	-	-	-	Motile bacillus	Penicillin G (Ampicillin), Sulfonamides Cephalosporin, Aminoglycoside
Proteus mirabilis	-	-	-	slant red (alkaline) & butt yellow (acidic), gas & H₂S produced	-	+	-	+	+	Highly motile bacillus	Trimethoprim-sulfamethoxazole, Ampicillin
Klebsiella pneumoniae	-	-	+	slant & butt yellow (acidic), gas produced, H₂S negative	-	-	+	+	+	Non-motile bacillus	Penicillin, Cephalosporin, Aminoglycoside, Cephalosporin (e.g. Cefotaxime)
Enterobacter spp	-	-	+	slant & butt yellow (acidic), gas produced, H₂S negative	-	-	+	+	-	Motile bacillus	Ciprofloxacin, Aminoglycoside (e.g. Gentamycin)
Pseudomonas aeriginosa	-	+	-	slant & butt red(alkaline), gas & H₂S negative	+	-	-	+	-	Motile bacillus	Penicillin, Cephalosporin, Aminoglycoside, Tetramycin

Table 1.1 Biochemical,Microscopy and Antibiotic Susceptibility Tests Results of Gram-Negative Microbes

Fig1.1 Left indicates negative Fig 1.2 Left indicates negative Methyl-red
Indole result, right is positive result, right is positive

Taken from http://www.google.com/>images>search>indole tests)
Taken from http://www.google.com/>image>search>methyl-red test)

Fig1.3 Left indicates negative Voges- Fig1.4 Fig1.1 Left indicates positive citrate

Proskauer result, right is positive result, right is negative

Taken from http://www.google.com/>image>search>Voges-Proskauer test

Taken from http://www.google.com/>image>search>Citrate test

	Gram Stain	Catalase	Coagulase	Haemolysis on Blood Agar	Wet Mount	Antibiotic Susceptibility Test
Staphylococcus saprophytcus	+	+	-	Non-hemolytic	Non-motile cocci	Norfloxacin, Trimethoprim-sulfamethoxazole (e.g. Bactrim)
Staphylococcus aureus	+	+	+	Beta hemolytic colonies	Non-motile cocci	Vancomycin, Amoxicillin with clavulanic acid, Cloxacillin, Ampicillin, Cephalosporon
Enterococcus faecalis	+	-		Non-hemolytic colonies (24 hours) Alpha-hemolytic(48 hours)	Non-motile cocci	Ampicillin, Vancomycin, Aminoglycoside

Table 1.2 Biochemical,Microscopy and Antibiotic Susceptibility Tests Results of Gram-positive Microbes

Reference

1. http://www.cst.cmich.edu>Urinary Tract Infections and Sexually Transmitted Infections

2. http://merck.com>search>fungal urinary tract infections

3. Warren Levinson Review of Medical Mrcrobiology and Immunolgy Ninth Edition

Case 2 (Azhar)

Patient: Kwan Siew Yan, Female, 29, outpatient

Complaints: Diarrhea

Diagnosis: Enterocolitis

Salmonella spp.

Commonly used agars used for culturing:

Differential media:

Eosin methylene blue (EMB) agar (growth of colourless colonies)

MacConkey’s agar (growth of colourless colonies)

Bismuth sulfite agar (growth of grey-brown or black colonies, they may or may not have a metallic sheen)

Selective media:

Salmonella-Shigella (SS) agar (growth of black colonies)

Hektoen enteric (HE) agar (growth of blue-green to blue colonies with black deposits in the middle of the colonies)

Xylose-lisine-deoxycholate (XLD) agar (growth of red colonies, they may or may not have black centres)

Biochemical Tests:

Indole negative (does not produce indole from tryptophan)

Citrate positive (able to utilize citrate)

Urease negative (does not produce urease)

Antimicrobial Sensitivity Testing:

Ampicillin - Susceptible

Ciprofloxacin - Susceptible

Chloramphenicol - Susceptible

Gentamycin - Susceptible

Microscopy:

Wet mount (Motile rod)

Gram stain (Gram-negative)

Shigella spp.

Commonly used agars used for culturing:

Differential media:

EMB agar (growth of colourless colonies)

MacConkey’s agar (growth of colourless colonies)

Selective media:

SS agar (growth of colourless colonies)

Hektoen enteric (HE) agar (growth of blue-green to blue colonies)

Xylose-lisine-deoxycholate (XLD) agar (growth of red colonies)

Biochemical Tests:

Indole negative (does not produce indole from tryptophan)

Citrate negative (unable to utilize citrate)

Urease negative (does not produce urease)

Antimicrobial Sensitivity Testing:

Cefuroxime - Susceptible

Chloramphenicol - Susceptible

Gentamycin - Susceptible

Microscopy:

Wet mount (Non-motile rod)

Gram stain (Gram-negative rod)

Campylobacter spp.

Commonly used agars used for culturing:

Differential media:

MacConkey’s agar (no growth)

Selective media:

Campylobacter Selective medium (grey to colourless colonies depending on species)

*incubation in microaerophilic condition (reduced oxygen [5%] and 10% carbon dioxide condition)

Biochemical Tests:

Catalase positive (contains catalase which converts hydrogen peroxide to water and oxygen)

Oxidase positive (contains cytochrome c oxidase)

Antimicrobial Sensitivity Testing:

Chloramphenicol - Susceptible

Gentamycin - Susceptible

Erythromycin - Susceptible

Microscopy:

Gram stain (Gram-negative spiral rods)

Wet mount (motile spiral rods)

Clostridium difficile

Commonly used agars used for culturing:

Differential media:

Blood agar plate (presence of haemolysis)

No biochemical tests available

Other tests:

Immunoenzymatic assay to detect toxins (either toxin A or B)

Antimicrobial Sensitivity Testing:

Ampicillin - Susceptible

Ceftazidime - Susceptible

Microscopy:

Gram stain (Gram-positive rods with a bulge on their terminal ends)

Vibrio spp

Commonly used agars used for culturing:

Differential media:

Blood agar plate (non-haemolytic colonies with a greenish hue)

Selective media:

Thiosulfate-citrate-bile-sucrose (TCBS) agar (growth of yellow colonies)

Biochemical Tests:

Oxidase positive (contains cytochrome c oxidase)

Lysine decarboxylase positive (able to decarboxylate lysine)

Arginine dihydrolase positive (converts arginine to citrulline and finally citrulline to ornithine)

Antimicrobial Sensitivity Testing:

Gentamycin - Susceptible

Ampicillin – Susceptible

Penicillin G - Resistant

Microscopy:

Gram stain (Gram-negative rods)

Wet mount (motile curved rods)

Escherichia coli

Commonly used agars used for culturing:

Differential media:

EMB agar (growth of purple colonies with a characteristic green sheen)

MacConkey’s agar (growth of red colonies)

Cysteine Lactose Electrolyte Deficient (CLED) agar (growth of yellow colonies)

Biochemical Tests:

Indole positive (produces indole from tryptophan)

Lysine decarboxylase positive (able to decarboxylate lysine)

Antimicrobial Sensitivity Testing:

Ciprofloxacin - Susceptible

Microscopy:

Gram stain (Gram-negative rod)

Wet mount (motile rods)

Yersinia spp.

Commonly used agars used for culturing:

Differential media:

MacConkey’s agar (growth of small colourless colonies)

*poor growth

Differential Selective media:

Cefsulodin-irgasan-novobiocin (CIN) agar (growth of colonies with a deep red centre that has a transparent margin)

Antimicrobial Sensitivity Testing:

Ampicillin - Resistant

Chloramphenicol - Susceptible

Ciprofloxacin - Susceptible

Gentamycin – Susceptible

Imipenam - Susceptible

Biochemical Tests:

Urease positive (produces urease)

Oxidase negative (does not contain cytochrome c oxidase)

Microscopy:

Gram stain (Gram-negative rod)

Wet mount (non-motile rods at 37°C) *motile at 25°C

Parasites

Microscopy:

Wet mount (presence of either ova [eggs] or parasite)

Stool ova & cyst (presence of either ova [eggs] or parasite)

Case 3 (Ming Boon)

Patient: Maisy Hong, Female, 67, inpatient

Complaints: Fever, chills, bladder distension, on indwelling catheter

Diagnosis: UTI

From the list of common causative microorganisms in the previous post, these are the microorganisms that are the most probable causative agents for the UTI in this patient as they are the infections are nosocomial and associated with catheter. The following tests are required in order to differentiate between the organisms.

Escherichia coli

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under anaerobic conditions)

Blood agar: Gamma hemolysis

Eosin Methylene Blue agar: Colonies with metallic green sheen

MacConkey agar: Pink colonies

Biochemical tests

Indole test: Positive
Methyl Red (MR) test: Positive
Voges-Proskauer (VP) test: Negative
Simmon’s citrate test: Negative
Oxidase test: Negative
Urease: Negative

Antimicrobial testing

- 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

Pseudomonas aeruginosa

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under aerobic conditions as it is a strict aerobe, able to grow at 42^oC)

Blood agar: Beta-hemolysis

MacConkey agar: Colourless colonies

Ordinary nutrient agar: Blue-green colonies

TSI agar: Growth with typical metallic sheen

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Negative
Simmon’s citrate test: Positive
Catalase test: Positive
Oxidase test: Positive
- Able to distinguish between the Enterobacteriaceae family as all the bacteria in this family are oxidase negative

Antimicrobial testing

- Naturally resistant to a broad spectrum of antibiotics including sulfonamide, trimethoprim
- Susceptible to a combination of antipseudomonal penicillin (eg. ticarcillin, piperacillin) and aminoglycoside (eg. gentamicin, amikacin)

Proteus mirabilis

Gram stain: Gram negative bacillus

Wet mount: Highly motile

Culture
(Culture under anaerobic conditions)

Blood agar with phenylethyl alcohol: Colonies do not have swarming effect

Colonies of P. mirabilis have a swarming effect, making it difficult to obtain pure isolates of other species. Addition of phenylethyl alcohol inhibits the swarming.

Picture taken from http://www2.bakersfieldcollege.edu/bio16/Lab%20Manual/Lab%20WEb%20Exercises/lab_exercise3.htm

MacConkey agar: Colourless colonies

TSI agar: Black butt

Biochemical tests

Indole test: Negative
Methyl Red test: Positive
Voges-Prokauer test: Negative
Simmon’s citrate test: Positive
Catalase test: Positive
Urease test: Positive
- Differentiate Proteus species from other members of Enterobacteriaceae

Antimicrobial testing

- Less resistant to antibiotics compared to the indole positive species
- Sensitive to ampicillin, aminoglycosides and trimethoprim-sulfamethoxazole

Enterobacter cloacae

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under anaerobic conditions)

MacConkey agar: Pink colonies

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Urease test: Positive

Antimicrobial testing

- Antibiotic resistance vary greatly
- Isolates from nosocomial infections are frequently resistant to multiple antibiotics
- Susceptible to aminoglycoside (eg. gentamicin) and cephalosporin (eg. cefotaxime)
- In severe infections, a combination of imipenem and gentamicin is required

Serratia marcescens

Gram stain: Gram negative bacillus

Wet mount: Motile

Culture
(Under anaerobic conditions)

MacConkey agar: Pink colonies (late lactose-fermenter so colonies might be colourless)

Ordinary nutrient agar: Red colonies

Picture taken from http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displayarticle&art_id=123

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Urease test: Negative

Antimicrobial testing

Klebsiella pneumoniae

Gram stain: Gram negative bacillus, a large capsule can be observed

Wet mount: Non-motile

Culture
(Under anaerobic conditions)

MacConkey agar: Pink colonies with mucoid appearance

Biochemical tests

Indole test: Negative
Methyl Red test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Urease test: Positive

Antimicrobial testing

Enterococcus faecalis

Gram stain: Gram positive cocci, in chains

Wet mount: Non-motile

Culture
(Under anaerobic conditions)

Blood agar: Gamma hemolysis

MacConkey agar: Pink colonies with mucoid appearance

Bile Esculin Agar: Ferric citrate indicator will turn black

Biochemical tests

Indole test: Negative
Voges-Prokauer test: Positive
Simmon’s citrate test: Positive
Catalase test: Negative

Antimicrobial testing

- Resistant to aminoglycoside, penicillin and vancomycin when given individually
- Requires a combination of aminoglycoside (eg. gentamicin) and penicillin or vancomycin
-Linezolid can be used against vancomycin-resistant enterococci

References:

1. Levinson W. 2006. Review of Medical Microbiology and Immunology, 9^th edition. McGraw Hill Companies
2. http://en.wikipedia.org>Pseudomonas
3. http://www.bact.wisc.edu>Microbiology Web Textbook
4. http://gsbs.utmb.edu>Current Students>Medical Microbiology>Chapter 27
5. http://en.wikipedia.org>Proteus mirabilis
6. http://www2.truman.edu/~kes251>Proteus mirabilis
7. http://gsbs.utmb.edu>Current Students>Medical Microbiology>Chapter 26

Case 4 (Michelle)
Name: Tong Wei Hong (Outpatient)
Sex: Male
Age: 68 years old
Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis

Streptococcus pneumoniae: The bacterium is a gram-positive coccus, lancet shaped, 0.5-1.2um in diameter that firms short chains or more commonly in pairs, hence by gram staining test, we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium does not have the catalase enzyme thus a negative result is expected when a biochemical identification test to detect the presence of catalase is carried out.
Colonies of this bacterium are alpha-hemolytic on blood agar when grown aerobically and beta-hemolytic when grown anaerobiaclly. The colonies are bile-soluble in which they are lysed in the presence of bile and their growth is inhibited by optochin. Hence this bacterium is optochin sensitive and it will soloubilise in bile. Blood cultures are positive in 15-25% of pneumoncoccal infections.
Antibiotic susceptibility test using penicillin, erythromycin, cephalosporin, chloramphenicol, vancomycin can be conducted. Penicillin has been the drug of choice against this bacterium though penicillin-resistant strains have been developing,thus vancomycin is used for these increasingly penicillin-resistant strains.
Additional serology tests such as Quellung reaction can be used to confirm the presence of pneumoncocci in gram stains of sputum smears in which anticapsular antibodies will cause the capsule polysaccharide of this bacterium to swell when viewed under the light microscope.

Haemophilus influenza: The bacterium is a small gram-negative rod (coccobacillus) with a polysaccharide capsule. Serologic typing is based on the antigenicity of the capsular polysaccharide in which of the six serotypes, type b causes most of the severe, invasive diseases hence by gram staining test we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium contains the enzyme oxidase thus a positive result is expected when a biochemical identification test to detect the presence of oxidase is carried out.
They are pleomorphic bacilli that require heme (factor X) and NAD+(factor V) for growth on heated-blood (“chocolate”) agar enriched with these growth factors. The blood is heated to inactivate non-specific inhibitors of the growth of this bacterium.
Antibiotic susceptibility test using ceftriaxone, trimethoprim-sulfamethoxazole, amoxicillin-clavulanate can be conducted. Penicillin is not effective against it.
Definitive identification can be made by serology testing by capsular swelling (Quellung) reaction in which the polysaccharide capsule of this bacterium will swell when viewed under the light microscope. Additional means of identifying encapsulated strains include fluorescent-antibody staining of the organism or latex agglutination test.

Moraxella catarrhalis: This bacterium is a gram-negative rod (coccobacillus) hence by gram staining test we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium contains the enzyme oxidase thus a positive result is expected when a biochemical identification test to detect the presence of oxidase is carried out.
Antibiotic susceptibility test using cephalosporins, erythromycin, tetracycline can be conducted.

Mycoplasma pneumoniae: It is a very small, free-living bacterium, in the class of Mollicutes. Many are as small as 0.3um in diameter and their most striking feature is the absence of a cell wall. They stain poorly with gram stain hence diagnosis is usually not by culturing sputum samples as it will take at least 1 week for colonies to appear on special media. Culture on regular media reveals only normal flora.
Serologic testing is the mainstay of diagnosis. A cold-agglutinin titer of 1:128 or higher is an indication of recent infection. However, the test is non-specific and false positive results occur in influenza virus and adenovirus infections.
The diagnosis of this bacterium can be confirmed by a 4-fold or greater in specific antibody titer in the complement fixation test.

Chlamydia pneumoniae: They are obligate intracellular bacteria. They lack the ability to produce sufficient energy to grow independently and therefore can grow only inside host cells. They are able to form cytoplasmic inclusions which can be seen by Giemsa stain or by immunofluorescence. Gram stain is not useful in this case.
They can be grown in cell cultures treated with cycloheximide which inhibits host cell but not chlamydial protein synthesis hence enhancing the bacterium replication. The bacterium form inclusions that do not contain glycogen in which glycogen-filled inclusions are visualised by staining with iodine.
Antibiotic susceptibility test using tetracycline such as doxycycline and macrolides such as erythromycin can be conducted.

Bordetella pertussis: It is a small, coccobacillary, encapsulated gram-negative rod, hence by gram staining test we are able to identify these colony characteristics for this bacterium under a light microscope.
This bacterium contains the enzyme oxidase thus a positive result is expected when a biochemical identification test to detect the presence of oxidase is carried out.
Additional identification test such as culturing the bacterium in Bordet-Gengou medium (potato-blood-glycerol) can be conducted. Bordetella pertussis produces small, domed, glistening colonies that resemble bisected pearls. The colonies are
usually surrounded by a zone of hemolysis; however, some strains of B. pertussis are not hemolytic.
Identification can be made also by agglutination with specific antiserum or direct fluorescent-antibody staining.

Case 5 (Yvonne)

Patient: Wong Fei Hong, Male, 37, outpatient

Complaints: Fever, swelling around the operation wound

Diagnosis: Wound infection

Introduction of SSI (Surgical Site Infection)
A wound is the result of physical disruption of the integument or the mucous membrane due to external trauma, or some mechanical agency. The establishment of infection is caused by the presence of bacterial pathogens within internal tissues. Wound infection can be classified under traumatic open-wound infection which is caused by trauma, and surgical-wound infection, acquired surgically or via intravenous medical devices. The latter category would be the investigation of interest for the case study as one of the symptoms shown was swelling around the operation wound.

There are conditions that are more effective than the choice of antimicrobial prophylaxis to minimize microorganism invasion during surgery, such as:

The skill of the surgeon and his ability to prevent the formation of fluid collections

- Limiting the extent of residual devitalized tissue

- Prompt removal of drains and devices that traverse and potentially injure the primary mucocutaneous barrier

Therefore, the risk of surgical wound infections is determined by technical problems with the operation, particularly bleeding, the amount of devitalized tissue created, and the need for drains within the wound, as well as such metabolic factors as obesity and diabetes. The environment plays an important role too. In the study carry out by Rubin¹, Staphylococcus aureus and Streptococcus pyogenes were found to be particularly virulent in surgical-acquired infection.

Definition of SSIs According to the National Nosocomial Surveillance Infections:

- Superficial Incisional SSI*

Occurs within 30 days after the operation;
Involves only the skin or subcutaneous tissue; and
At least 1 of the following:

Purulent drainage (culture documentation not required)
Organisms isolated from fluid/tissue of superficial incision
At least 1 sign of inflammation (eg, pain or tenderness, induration, erythema, local warmth of the wound)
Wound is deliberately opened by the surgeon
Surgeon or attending physician declares the wound infected.

*A wound is not considered a superficial site infection if a stitch abscess is present, the infection is at an episiotomy or circumcision site or a burn wound, or the SSI extends into the fascia or muscle.

- Deep Incisional SSIs

Occurs within 30 days of operation or within 1 year if an implant is present;
Involves deep soft tissues (eg, fascia and/or muscle) of the incision; and
At least 1 of the following:

Purulent drainage from the deep incision but without organ/space involvement
Fascial dehiscence or fascia is deliberately separated by the surgeon due to signs of inflammation
Deep abscess is identified by direct examination or during reoperation, by histopathology, or by radiologic examination
Surgeon or attending physician declares that deep incisional infection is present.

- Organ/Space SSI

Occurs within 30 days of operation or within 1 year if an implant is present;
Involves anatomic structures not opened or manipulated during the operation; and
At least 1 of the following:

Purulent drainage from a drain placed by a stab wound into the organ/space
Organisms isolated from organ/space by aseptic culturing technique
Identification of abscess in the organ/space by direct examination, during reoperation, or by histopathologic or radiologic examination
Diagnosis of organ/space SSI by surgeon or attending physician.

Source: National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 to June 2002, issued August 2002.

Centers for Disease Control, Atlanta, Georgia.
http://www.cdc.gov/ncidod/hip/NNIS/2002NNISReport_AJIC.PDF

As the site and the degree of infection were not stated in the case given, we are unable to determine the severity and the possible bacteria strains.

Pathogenesis of SSI

All surgical wounds involved bacteria invasion while only a minority demonstrates clinical infection. In most patients, infection does not develop because innate host defenses are quite efficient in the elimination of contaminants at the surgical site.

However for the case of Wong Fei Hong, fever and swelling of the operation wound was manifested. This indicates that inflammation have taken place.

Determinants of Infection

4 important determinants that leads to SSI: (1) inoculum of bacteria, (2) virulence of bacteria, (3) adjuvant effects of microenvironment, and (4) innate and acquired host defenses.

These are the list of suspected microorganisms involved in SSI:

1. Enterobacter spp. (gram-negative rod)

Reasons for selection:

- opportunistic pathogens (large intestine) that causes nosocomial infections

- related to hospitalization, especially to invasive procedure such as intravenous catheterization

Biochemical tests:

- MacConkey’s or EMB agar: lactose-fermenting colonies (colored)

- Triple sugar iron (TSI) agar: Slant – acid; Butt – acid; Gas – (+); H₂S – (-)

Treatment (antibiotic susceptibility):

- Imipenem: 95%

- Netilmicin: 93%

- Ciprofloxacin: 90%

In severe Enterobacter infection, a combination of imipenem and gentamicin is often used.

2. Escherichia coli (gram-negative rods)

Reasons for selection:

- most abundant facultative anaerobe in the colon

- Endotoxin released causes fever

- Most frequent cause of nosocomial infections due to indwelling urinary catheters

Biochemical tests:

- Triple sugar iron (TSI) agar: Slant – acid; Butt – acid; Gas – (+); H₂S – (-)

- Blood agar

- EMB agar: green sheen colonies

Other tests:

- Presence of indole from tryptophan

- Microscopy: motile

Treatments (antibiotic susceptibility):

span >- Ceftriaxone: 100%

- Imipenem: 100%

- Ampicillin: 66%

3. Klebsiella spp.(gram-negative rods)

Reasons for selection:

- Opportunistic pathogens that cause nosocomial infections, especially pneumonia

Biochemical tests:

- Triple sugar iron (TSI) agar: Slant – acid; Butt – acid; Gas – (+); H₂S – (-)

- MacConkey’s or EMB agar: lactose-fermenting colonies (colored)

Treatment (antibiotic susceptibility):

- Ceftriaxone: 100%

- Imipenem: 100%

- Netilmicin: 100%

4. Proteus spp. (gram-negative rods)

Reasons for selection:

- Hospital-acquired infection, causing urinary tract infection

- Present in human colon

Biochemical tests:

- Urease test

- Blood agar: “swarming” overgrowth

- MacConkey’s or EMB agar: non-lactose fermenting (colorless)

- Triple sugar iron (TSI) agar: results varies in different species

Other test:

- Presence of enzyme phenylalanine deaminase

Treatment (antibiotic susceptibility):

- AMX-CLV (amoxicillin-clavulanate): 100%

- Ceftriaxone: 100%

- Imipenem: 100%

5. Serratia spp. (gram-negative rods)

Reasons for selection:

- Opportunistic pathogens that cause nosocomial infection, pneumonia

- Due to invasive procedures such as intravenous catheterization and respiratory intubation

Biochemical tests:

- Triple sugar iron (TSI) agar: Slant – alkaline; Butt – acid; Gas – (-); H₂S – (-)

- MacConkey’s or EMB agar: late lactose fermenters (S. marcescens produces red-pigmented colonies)

Treatment (antibiotic susceptibility):

- Imipenem: 96%

- Ceftriaxone: 92%

- Netilmicin: 94%

6. Pseudomonas aeruginosa (gram-negative rods)

Reasons for selection:

- Able to withstand disinfectants; hospital-acquired infection

- Found growing in hexachlorophene-containing soap solutions, in antiseptics.

- Colonizes the upper respiratory tract of hospitalized patients

- Contaminates anesthesia equipment, intravenous fluids

- Opportunistic pathogens, most common cause of gram-negative nosocomial pneumonia

Biochemical tests:

- MacConkey’s or EMB agar: non-lactose fermenting (colorless)

- Oxidase test: positive

- Triple sugar iron (TSI) agar: Slant – alkaline; Butt – alkaline; Gas – (-); H₂S – (-)

- Ordinary nutrient agar: blue-green pigment

Treatment (antibiotic susceptibility):

- Imipenem: 89%

- Ceftazidime: 87%

- Netilmicin: 72%

7. Acinetobacter spp. (gram-negative coccobacillary rods) – minor bacterial pathogens

Reasons for selection:

- Opportunistic pathogens that readily colonize patients with compromised immune system

- Causes disease in a hospital setting, associated with respiratory therapy equipment and indwelling catheters

Biochemical test:

- Gram stain

Treatment (antibiotic susceptibility):

- Imipenem: 95.2%

- Ciprofloxacin: 92.8%

- Netilmicin: 92.8%

8. Staphylococcus spp. / S. aureus (gram-positive cocci)

Reasons for selection:

- Most common microorganism present in surgical-wound infection

- Often found in the nose and skin in hospital staff and patients

- Affects individuals with compromised immune system

- A major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices

- Localized host response to staphylococcal infection is inflammation, characterized by fever and swelling

Biochemical tests (S. aureus):

- Coagulase test: positive

- Mannitol-salt agar: golden-yellow colonies, beta-hemolytic

Biochemical tests (Staphylococcus spp. other than S. aureus)

- Coagulase test: negative

- White colonies, non-hemolytic

Other tests:

- Smears reveal gram-positive cocci in grapelike clusters

Treatment (antibiotic susceptibility):

MS (methicillin-susceptible) Staphylococcus aureus

- AMX-CLV (amoxicillin-clavulanate): 100%

- Teicoplanin: 100%

- Vancomycin: 100%

MR (methicillin-resistant) Staphylococcus aureus

- Teicoplanin: 100%

- Vancomycin: 99.1%

- Netilmicin: 90.4%

MS (methicillin-susceptible) coagulase-negative staphylococci

- Imipenem: 100%

- Teicoplanin: 100%

- Vancomycin: 100%

MR (methicillin-resistant) coagulase-negative staphylococci

- Teicoplanin: 98.6%

- Vancomycin: 98.6%

- Netilmicin: 91.5%

9. Enterococcus faecalis (gram-positive cocci)

Reasons for selection:

- Important cause of hospital-acquired urinary tract infections and endocarditis

- Indwelling urinary catheters and urinary tract instrumentation are frequent predisposing factors

- Cause intra-abdominal and pelvic infections, in combination with anaerobes

Biochemical tests:

- Catalase test: negative

- Blood agar: alpha-, beta-, or non-hemolytic

Other tests:

- Grows in 6.5% NaCl

- Hydrolyzes esculin in the presence of 40% bile

Treatment (antibiotic susceptibility):

- Imipenem: 100%

- Teicoplanin: 97.9%

- AMX-CLV (amoxicillin-clavulanate): 72.9%

VRE (vancomycin-resistant enterococci) are important causes of nosocomial infections. Linezolid would be the drug of choice for the treatment of VRE.

10. Bacteroides fragilis (gram-negative rods)

Reasons for selection:

- Caused a variety of infectio

- Usually arise from a break in a mucosal surface

- Predisposing factor such as surgery play an important role in pathogenesis

- Associated with intra-abdominal infections

Biochemical tests:

- Sugar fermentation test

- Blood agar

- Organic acid tests

Treatment:

- Metronidazole

- mnioglycosides: treatment of facultative gram-negative rods in mixed infections

11. Clostridium perfrigens (gram-positive rods)

Reasons for selection:

- Common open-wound infection

- Edema occur in the wound area

- A small inoculum to cause an especially severe necrotizing infection at the surgical site

Biochemical tests:

- Sugar fermentation test

- Organic acid tests

- Blood agar: double zone of hemolysis

- Egg yolk agar: presence of lecithinase

Treatment:

- Penicillin G is the antibiotic of choice

Other causative agents that cause wound infection, such as Pasturella multocida which are transmitted by animals and Wuchereria bancrofti, transmitted by mosquitoes primarily in Africa, were ruled out in this investigation.

From the various study, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterocuccus faecalis are some of the pathogens commonly observed in surgical wound infection.

Case 6 (Shahirah)

Patient's name: Ong Fei Fei

The following agents are the most likely causative agent in this patient.

Proteus mirabilis

Gram stain: gram-negative bacillus
Wet mount: motile (highly)
Biochemical Tests:
Indole test: negative
Methyl Red test: positive
Voges-Prokauer tests: negative
Simmon’s citrate test: positive
Urease test: positive
Catalase test: positive
Culture: (under anaerobic conditions)
Blood agar: non-hemolytic
MacConkey agar: Colourless colonies
TSI agar: Black butt
Antimicrobial susceptibility: Ampicilin, aminoglycoside

Pseudomonas aerugionsa
Gram stain: gram-negative rods
Wet mount: Non-motile
Biochemical Tests:
Indole test: negative
Methyl Red test: negative
Voges-Prokauer test: negative
Simmon’s citrate test: positive
Oxidase test: positive
Catalase test: positive
Culture: (aerobic condition)
Blood agar: B-hemolytic
MacConkey agar: Colourless colonies
TSI agar: Negative
Antimicrobial susceptibility: gentamicin, cefepime, ciprofloxacin

Trichomonas vaginalis
Gram stain: Not useful as microorganism is a protozoa
Wet mount: Motile (jerky motion, corkscrew motility)
Culture: Best cultured at 35-37oC, anaerobically , pH 5.5-6.0
Antimicrobial susceptibility: metronidazole

Staphylococcus aureus
Gram stain: gram- positive cocci, cluster (similar to grapes)
Wet mount: Non-motile
Biochemical tests:
Catalase test: positive
Coagulase test: positive
Culture: (aerobic conditions)
Mannitol salt agar: Agar turns yellow due to the acid produced
Blood agar: B-hemolytic
Antimicrobial susceptibility: vancomycin, nafcillin

Staphylococcus saprophyticus
Gram stain: gram-positive cocci
Wet mount: non-motile
Biochemical Tests:
Catalase test: positive
Coagulase: negative
Urease: positive

Culture:
· Blood agar: gamma-hemolytic
Antimicrobial susceptibility: Quinolones

Eschericia coli
Gram stain: gram-negative rods
Wet mount: Highly motile
Biochemical Tests:
Indole test: Positive
Methyl Red test: Positive
Voges-Prokauer test: Negative
Simmon’s citrate test: Negative
Oxidase test: negative
MUG test: positive
Culture:
Blood agar (hemolysis)
MacConkey agar: Pink/red colonies
Antimicrobial susceptibility: amoxicillin, nitrofurantoin, trimethoprim-sulfamethoxazole

Gardnerella vaginalis
Gram stain: gram-variable rods
Wet mount: clue cells
Biochemical Tests:
Catalase test: negative
Culture:
Starch agar with bromcresol blue
Chocolate agar
Antimicrobial susceptibility: metronidazole

References: http://www.asmpress.org/asmpress/files/ccLibraryFiles/Filename/000000000713/CMPH2_2007_update_Section_3_for_e-store.pdf

http://www.emedicine.com/med/topic841.htm
http://www.textbookofbacteriology.net/staph.html
Brooks, Geo F., Janet Butel, and Stephen Morse. Jawetz, Melnick, and Adelberg's Medical Microbiology, 23rd edition. 2004.
Levinson, Warren. Review of Medical Microbiology and Immunology, Ninth edition. 2006.

MMIC Blog Post 1

2007-12-02T05:32:00.000-08:00

Case One

Patient: Khong Fay Seah, Female, 27, outpatient

Complaints: Fever, chills, dysuria

Diagnosis: Urinary Tract Infection (UTI)

The urinary tract includes the kidneys, ureters, bladder and urethra. As compared to men and children, women are of greater risk in acquiring urinary tract infection due to the shorter urethra and close proximity between the anus, urethra and vagina.

UTI is a common infection that occurs when bacteria enter the opening of the urethra and multiply in the urinary tract. Possible source of infections include the transfer of natural flora in fasces from the anus to urethra due to poor hygiene practices and the travelling of enteric tract organism (e.g. E.coli) to the urethra colonization. UTI can also be sexually transmitted.

There are two classifications of UTI, mainly lower and upper. While lower UTI involves the bladder and urethra, upper UTI implicates the ureters and kidneys. The common symptoms of lower UTI includes back pain, blood in the urine (hematuria), cloudy urine, fever, polyuria, general discomfort (malaise) and painful urination (dysuria). As for upper UTI, it involves high fever, shaking chills, nausea and may or may not include symptoms of lower UTI.

Common Causative Organisms

Supporting reason(s)

Investigation(s)

Bacteria

Escherichia Coli

(Gram-negative bacillus)

Occurs in more than 70% of UTI cases

Present in human colon as nature flora and is present in feces

-Blood Agar

-EMB Agar (Green sheen colonies)

-MacConkey’s Agar (Pink colonies)

Staphylococcus saprophyticus and aureus

(Gram-positive cocci)

Causes community-acquired urinary tract infection in young woman

-Mannitol-salt Agar (Yellowish-gold colonies)

Enterococcus faecalis

(Gram-positive cocci)

Present in human colon as nature flora and is present in feces

Largely involved in lower UTI

-EMB Agar (Coloured colonies)

MacConkey’s Agar (Coloured colonies)

Proteus mirabilis

(Gram-negative bacillus)

Vigorously motile contributing to their ability to invade urinary tract

Present as normal flora in colon

-Phenylethyl alcohol containing blood agar (inhibit swarming)

-EMB (Colourless colonies)

-MacConkey’s agar

Klebsiella pneumoniae

(Gram negative bacillus)

Involves in nosocomial infections especially UTI

Common causative agent of patients with catheter

-EMB Agar (Coloured colonies)

MacConkey’s Agar (Coloured colonies)

Enterobacter spp.

(Gram-negative bacillus).

Frequently found in large intestine

EMB Agar (Coloured colonies)

-MacConkey’s Agar (Coloured colonies)

Pseudomonas aeruginosa

(Gram-negative bacillus)

Opportunistic pathogen

10% of human carry it as nature flora of colon

EMB Agar (Colourless colonies)

MacConkey’s Agar

Fungal

Candida albicans

Frequently infects people who have an impaired immune system or a bladder catheter in place.

Sabouraud’s Agar

Virus

Herpes Simplex Virus Type 2 (HSV-2)

Common causative agent of genital lesions

Proliferates in female genital tract

Identify by immunological tests

Protozoa

Trichomonas vaginalis

Transmitted via sexual activities

Causes yellow-green vaginal discharge with a strong odor

Wet mount examined for trichomonads

Fig 1.1 On the left shows the non-lactose
culture (colourless) and on the right lactose-fermenter (metallic green sheen) on EMB agar

(Taken from http://www.google.com/ > images > search >E.coli on EMB)

Fig1.2 Lactosing Fermenting organism forming pink
colonies on MacMonkey Agar

(Taken from http://www.google.com/ > images > search >E.coli on MacConkey)

Fig 1.3 Staphylococcus saprophycius on Mannitol-salt Agar

(Taken from http://www.google.com/ > images > search >Staphylococcus on Mannitol-salt Agar)

Though Trichomonas vaginalis and Herpes Simplex Virus Type 2 (HSV-2) lead to UTI, it mainly causes vaginitis instead which leads to vaginal discharge. These symptoms is not observed for Ms Khong Fay Seah Also, candidal urethritis is in healthy women unless catheter is in placed and thus we rule out Candida albicans infection.

Done by: Jiaxin

Case 2

Patient: Kwan Siew Yan, Female, 29, outpatient

Complaints: Diarrhea

Diagnosis: Enterocolitis

Enterocolitis is the inflammation of both the small and large intestines. Most of the time however, it is categorized as either of the following:

Enteritis: which is the inflammation of the small intestine

Colitis: which is the inflammation of the large intestine, especially the co

A few types of enterocolitis can occur and these include:

Pseudomembranous enterocolitis

Thought to be caused by a toxin-producing strain of Clostridium difficile

Necrotic mucosa is replaced by a pseudomembrane filled with staphylococci, leukocytes, mucus, fibrin, and inflammatory cells

Has occurred postoperatively in debilitated patients who has undergone abdominal surgery and in patients treated with broad-spectrum antibiotics (Ampicillin, clindamycin, and cephalosporins are suspected as causative factors)

Immunocompromised patients are at increased risk for this disease

Salmonella enterocolitis

An infection in the lining of the small intestine caused by Salmonella

It occurs when food or water that is contaminated with Salmonella are ingested

Enterocolitis caused by parasites

Enterocolitis caused by medications

Necrotizing enterocolitis (NEC)

Usually occurs in premature neonates (less than 34 weeks’ gestation) and those of low birth weight (less than 5 lb [2.3 kg]). NEC is occurring in more neonates, possibly because of the higher incidence and survival of premature and low-birth-weight neonates

Since it is most commonly seen in neonates and premature and low-birth-weight neonates, the patient is probably not suffering from this type of enterocolitis

Symptoms include pain, swelling, cramping or tenderness of the abdominal area, diarrhoea, nausea, vomiting, fever, chills rectal bleeding, sluggishness, and muscle pain

Stool(faecal) sample

Screening of faeces should be routine for Salmonella, Shigella, Compylobacter and Vibrio spp. The role of Aeromonas and Plesiomonas spp. in causing diarrhoea is controversial and these are reported only when found in pure and heavy growth. On occasions, enteropathogenic E. coli (EPEC) cause problems in neonatal outbreaks and like other diarrhoegenic E. coli (ETEC, EIEC and EAEC), they are not readily detected by current reagents and media. Where indicated, Yersinia spp., enterohaemorrhagic E. coli and C. difficile (cause of hospital-associated or antibiotic-associated diarrhoea) may be cultured.

Common Causative Organisms	Supporting reasons	Investigations
Salmonella spp. (Gram-negative)	- Certain strains of Salmonella can infect the lining of the small intestine causing Salmonella enterocolitis	Culture on: - Salmonella- Shigella (SS) plate - BAP - MAC - Campylobacter Selective Medium - Thiosulphate-citrate bile sucrose agar (TCBS) - Selenite F broth - Alkaline Peptone Water (APW) - Enterococcosel Agar with 6ug/mL of vancomycin - Sorbitol MAC for enterohaemorrhagic E. coli All are grown under aerobic conditions except for Campylobacter selective medium which requires anaerobic incubation Microscopy: - Gram stain - Wet mount - Parasite - Stool ova & cyst
Shigella spp. (Gram-negative)	- Causes dysentery (severe diarrhoea) that result in the destruction of the epithelial cells of the intestinal mucosa in the cecum and rectum - Symptoms include diarrhea, fever, nausea, vomiting, stomach cramps and the stool may contain blood, mucus, or pus
Campylobacter spp. (Gram-negative)	- Causes campylobacteriosis, an inflammatory, sometimes bloody, diarrhea or dysentery syndrome, mostly including cramps, fever and pain
Clostridium difficile (Gram-positive)	- Toxins produced by this organism are thought to cause Pseudomembranous enterocolitis
Vibrio spp (Gram-negative)	- Commonly found in seafood and may cause gastroenteritis (diarrhoea)
- Enteropathogenic Escherichia coli [EPEC](Gram-negative) - Enterohaemorrhagic E. coli - Diarrhoegenic E. coli [ETEC, EIEC and EAEC] - Yersinia spp. (Gram-negative)	- These organisms are possible causative agents of enterocolitis
Parasites	- The presence of parasites may cause irritation and inflammation of the intestines and thus causing diarrhoea

Done by: Azhar

Case 3

Patient: Maisy Hong, Female, 67, inpatient

Complaints: Fever, chills, bladder distension, on indwelling catheter

Diagnosis: UTI

As the patient is suffering from bladder distension, a condition in which she is unable to urinate by herself, an indwelling catheter is used. The use of the indwelling catheter is an important predisposing risk factor for UTI as bacteria from the anus can be easily passed up the urethra because of the location of the insertion. Furthermore, presence of bacteria within the catheter can occur if it is not sanitised properly. As the patient is hospitalised, this is probably a nosocomial (hospital-acquired) infection.

The patient might be suffering from lower UTI as the symptoms (fever and chills) are often observed in lower UTI. In upper UTI, the symptoms are usually more serious (high fever and shaking chills). However, infections associated with catheters usually do not have symptoms such as dysuria, increased frequency and urgency to urinate, suprarubic and back pain so it is difficult to confirm the diagnosis.

As the patient is on indwelling catheter, it can be assumed that the urine sample is collected through the catheter.

Common Causative Organisms	Supporting reasons	Investigations
Escherichia coli (Gram-negative)	- Predominant causative agent of UTI - Most frequent cause of nosocomial UTI and are associated with the use of indwelling catheters	Culture on: BAP EMB agar MAC Ordinary nutrient agar Triple Sugar Iron (TSI) agar All are grown under anaerobic conditions except for P. aeruginosa as it is a strict aerobe Microscopy Gram stain Fungal stain
Pseudomonas aeruginosa (Gram-negative)	- Causes infection in almost anywhere in the body but main infections include UTI - Infections occur primarily in patients who are immunocompromised and are associated with the use of indwelling catheters
Enterococcus faecalis (Gram-positive)	- Causes UTI especially in hospitalized patients - Use of indwelling catheter is an important predisposing factor
Klebsiella pneumoniae (Gram-negative)	- Emerging as an important nosocomial pathogen - Causes UTI that are catheter-associated but most infections are lungs-associated
Enterobacter cloacae (Gram-negative)	- Important nosocomial pathogens that cause a variety of infections including UTI - Especially in patients of extreme ages and associated with the use of indwelling catheter
Serratia marcescens (Gram-negative)	- Causes UTI in hospitalized patients associated with the use of catheter
Proteus mirabilis (Gram-negative)	- Once attached to the urinary tract, it infects the kidney more commonly than E. coli. - Its vigorous motility contributes to its ability to invade the urinary tract

Staphylococcus saprophyticus, Candida albicans, Adenovirus also causes UTI. UTI caused by S. saprophyticus is particularly in sexually active young woman so it is not possible in this case. Candida albicans might not be the causative agent in this case because the patient does not have symptoms such as itching and vaginal discharge that are seen in vaginitis (infection by Candida). Adenovirus (Types 11 and 21) causes hemorrhagic cystitis. It is also not a possible causative agent because prominent symptoms such as hematuria and dysuria are not observed in the patient.

Done by: Ming Boon

Case 4

Name: Tong Wei Hong, Male, 68, outpatient

Complaints: Fever, chills, excessive phlegm, breathing problems

Diagnosis: Bronchitis

Bronchitis is an inflammation of the bronchial tubes, or bronchi, that bring air into the lungs. It can either be acute or chronic. In acute bronchitis, it usually begins with symptoms of cold such as runny nose, slight fever and dry cough. If remain unhealed, it could slowly progress to chronic bronchitis in which the person will have difficulties in breathing, coughing with phlegm. If the disease advances, wheezing will occur and is accompanied with shortness of breath. Bronchitis is mostly due to viruses such as influenza A and B and those who have chronic medical problems may experience shortness of breath while having the flu. Bronchitis due to viral infections can be complicated with bacterial infections and this can be observed in the colour of the phlegm. With viral bronchitis, small amounts of white mucus are often coughed up. When the coughed-up mucus changes from white to green or yellow, the condition might be complicated by a bacterial infection.

Mycoplasma pneumoniae and Chlamydia pneumoniae often cause bacterial bronchitis in young adults. Among middle-aged and older people, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most common organisms causing bacterial bronchitis. Bordetella pertussis could also be another possible causative agent for bronchitis as it is responsible for pertussis, commonly known as whopping cough, which is an acute tracheobronchitis. However, pertussis is considered as a pediatric disease and is typically uncommon in adults.

A person often has a combination of bacterial and viral bronchitis. Hence the possible pathogenic bacteria responsible for bronchitis of this patient are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.

Possible causative agents. (Types of microorganisms)	Supporting reasons	Preliminary lab investigations
Streptococcus pneumoniae	These are the most common microorganisms that caused bacterial bronchitis in middle-aged and older people. Symptoms of bronchitis will be seen.	Gram staining test and identify colony characteristics under a light microscope. Biochemical identification test to detect the presence of catalase. Identify type of haemolysis on blood agar. Antibiotic susceptibility test using penicillin, erythromycin and vancomycin.
Haemophilus influenza		Gram staining test and identify colony characteristics under a light microscope. Biochemical identification test to detect the presence of oxidase. Antibiotic susceptibility test using Ceftriaxone, trimethoprim-sulfamethoxazole. Identify microorganism on heated blood agar enriched with factor X( a heme compound) and factor Y (NAD).
Moraxella catarrhalis		Gram staining test and identify colony characteristics under a light microscope. Biochemical identification test to detect the presence of oxidase. Antibiotic susceptibility test using erythromycin and tetracycline.
Mycoplasma pneumoniae	Mycoplasma pneumoniae and Chlamydia pneumoniae often cause bacterial bronchitis in young adults (5-15years old). Symptoms of bronchitis will be seen. For Mycoplasma pneumoniae, it will cause primary atypical pneumonia in which the body will responds with a persistent unproductive cough in an attempt to clear lungs. For Chlamydia pneumoniae, it causes 5% of cases of bronchitis and it usually cause atypical pneumonia.	Serologic testing using antibodies.
Chlamydia pneumoniae		Giemsa stain or by immunofluorescence identify colony characteristics. Perform cell cultures treated with cycloheximide. Antibiotic susceptibility test using tetracycline such as doxycycline and macrolides such as erythromycin can be conducted.
Bordetella pertussis	It is responsible for the disease (pertussis) commonly known as whooping cough which is a type of bronchitis. Symptoms include cough and excessive production of mucus.	Gram staining test and identify colony characteristics under a light microscope. Biochemical identification test to detect the presence of oxidase. Perform cell culture in Bordet-Gengou medium. Serologic testing using antibodies.

Done by: Michelle

Case 5

Patient: Wong Fei Hong, Male, 37, outpatient

Complaints: Fever, swelling around the operation wound

Diagnosis: Wound infection

As the given information stated that it was an operation wound, the likelihood of the patient suffering from surgical-wound infection would be higher, rather than infection caused by injury.

Common Causative Organisms	Supporting reasons	Investigations
Staphylococcus aureus (Gram-positive)	- Most common microorganism present in surgical-wound infection - A major cause of nosocomial infection of surgical wounds and infections associated with indwelling medical devices - The localized host response to staphylococcal infection is inflammation, characterized by fever and swelling	Culture on: anaerobic condition BAP MAC Mannitol-salt agar Microscopy: Gram stain
Pseudomonas aeruginosa (Gram-negative)	- Cause infections in the body, primarily wound infection due to burns - An opportunistic pathogen that causes infections in hospitalized patients (immunocompromised)
Bacteriodes fragilis (Gram-negative)	- Causes a variety of infections - Predisposing factor such as surgery play an important role in pathogenesis
Clostridium perfringens (Gram-positive)	- Common in open-wound infections - Edema occurs in the wound area

Case 6

Patient: Ong Fei Fei, Female, 37, outpatient

Complaints: Fever, pain during urination, vaginal discharge

Diagnosis: UTI

Common causative agents

Supporting reasons

Investigations

Eschericia coli

(gram-neg bacteria)

Normal flora of anterior

urethra as well as vagina Common agent in UTI

Culture on:

-BAP
-MAC

Microscopy:

-Fungal stain

-Wet mount

-Gram stain

Candida albicans

(fungus-yeast)

- Opportunistic pathogen which exist as normal flora in the vagina of some women

- Causes infection when normal flora of the genital tract is disrupted

- Usually seen in patients with indwelling catheter or on antibiotic treatment

Staphylococcus aureus

(gram-positive cocci)

- Found on mucosal lining of vagina

- Overgrowth may lead to infection of the urethra

Proteus mirabilis

(gram-neg bacteria)

- Found as normal flora in the genital tract

- Possible agent of UTI

Staphylococcus saprophyticus

(gram-positive cocci)

- 2^nd most common UTI agent in sexually active women

Trichomonas vaginalis

(protozoa)

-Causes trichomoniasis

-Greenish vaginal discharge is watery, foul-smelling.

Chlamydia trachomatis

(gram-neg)

- Obligate intracellular bacterium

- Cause urethritis in both male and female

Klebsiella pneumoniae

(gram-neg)

- Non-motile

- Causes UTI among other diseases

- Ubiquitous in the enviroment

Pseudomonas aeruoginosa

(gram-neg rod)

- Aerobic bacterium

- Motile

Serratia marcesens

(gram-neg rod)

- Facultative anaerobe

- Causes UTI

Gardnerella vaginalis

(gram- variable rod)

- Agent in bacterial vaginosis

- Vaginal discharge is pungent, fishy-odour

Mobiluncus sp.

(gram- +ve rod)

- Causative agent in bacterial vaginosis

- Anaerobic bacterium

Enterococcus faecalis

(gram- +ve cocci)

- Found in the colon

- Alpha-hemolytic

The above are the possible causative agents of UTI in this patient. Other possible agents like Ureaplasma ureatylticum and Mycoplasma genitalium are excluded as they cause urethritis in men only.

Done by: Shahirah

References:

1. http://www.emedicinehealth.com>urinary tract infection>page 3
2. http://www.merck.com>urinary tract infection
3. http://www.wrongdiagnosis.com>enterocolitis
4. http://www.nlm.nih.gov/medlineplus>salmonella enterocolitis
5. http://en.wikipedia.org/wiki/Main_Page>enterocolitis
6. http://www.wrongdiagnosis.com>bladder distension
7. http://www.niddk.nih.gov>overcoming bladder disease
8. http://www.cdc.gov>guideline for prevention of catheter-associated urinary tract infections
9. Levinson W. 2006. Review of Medical Microbiology and Immunology, 9th edition. McGraw Hill Companies
10. http://www.merck.com>bronchitis
11. http://respiratory-lung.health-cares.net
12. http://www.bact.wisc.edu>the microbial world>normal flora
13. http://www.columbia.edu>urinary tract infections
14. http://www.hmc.psu.edu>urinary tract infections
15. http://en.wikipedia.org/wiki/Main_Page>proteus mirabilis
16. http://www.searo.who.int/en>urinary tract infection
17. Brooks, Geo F, Butel J & Morse S. 2004. Jawetz, Melnick, and Adelberg's Medical Microbiology, 23rd edition

Sorry for the tables! Did my best already!

Lab Techniques

2007-11-10T05:52:00.001-08:00

Hi all, SIP/MP is finally over!!

Today I'm going to talk about the extraction of cell membrane proteins. Jiaxin had described how we extracted secretory proteins using TCA/Acetone precipitation method. For the extraction of cell membrane proteins, we are using ReadyPrepTM Protein Extraction Kit (Membrane I) from BIORAD.

Picture is taken from http://www.biocompare.com/images/spotlight/spot_isc205_biorad.jpg

Principle:

The membrane proteins are extracted based on temperature-dependent phase partitioning. The sample is first mixed and homogenized in the extraction buffers (buffer M1 and M2) provided in the kit. After a brief incubation at 37oC, the sample is centrifuged at 28oC to produce 2 phases. The top layer (hydrophilic) will contain cytoplasmic proteins, bottom phase (hydrophobic) will contain the membrane proteins and an insoluble pellet would contain the complex membrane proteins that are not solubilized by the extraction buffers.

The components of the extraction buffers are not revealed by the manufacturer and thus the exact mechanism is unknown.

Procedures:

The first 3 days are the same (Refer to Jiaxin's last post).

On the 4th day (Extraction of proteins using the kit)
- After the supernatant is used for extraction of secretory proteins, the cell pellet will be used for labelling with Cy3. Extraction of the proteins will only start after the labelling is done.

1. Centrifuge the tube at 5000xg for 20 min at 4oC (to pellet the cells)
2. Transfer the supernatant to a new 1.5mL eppendorf tube and store
3. Add an appropriate volume of buffer M1(0.5mL to 0.05mL of cell pellet) to the cell pellet and resuspend
4. Add an appropriate volume of protease inhibitor(1uL to 100uL of buffer M1) to the resuspended sample
5. The suspension was splited equally into 5 tubes (if not subsequently there will be not enough space to add buffer M2)
6. Sonicate the suspension for 35 sec (to lyse the cells)
7. Chill the suspension on ice for 1 min (sonication would produce heat)
8. Repeat steps 6 and 7 for 6 times
9. Add an appropriate volume of buffer M2(equal volume to buffer M1) to the suspension and mix well
10. Vortex the suspension for 1 min
11. Chill the suspension on ice for 1 min
12. Repeat steps 10 and 11 for 7 times
13. Chill the suspension on ice for 10 min
14. Transfer the suspension to 37oC water bath for 30 min (Invert tubes thrice for 30 sec to mix the suspension)
15. Centrifuge the tubes at 16,000xg for 5 min at 28oC
16. Transfer the top layer(containing hydrophilic proteins) to a new 2.2mL eppendorf tube and store
17. Add an appropriate volume of buffer M2(equal amount as step 9) to each of the bottom phase and mix well
18. Repeat steps 10 to 16
19. Transfer the bottom phase(containing the hydrophobic proteins) to a new 2.2mL eppendorf tube and store

The bottom phase which contains the hydrophobic proteins is our interest as most membrane proteins are hydrophobic.

That is all! Thanks=)

Chua Ming Boon
0503197F
TG01

Immunology/MMIC

2007-11-06T06:06:00.000-08:00

Procedure: Venereal Disease Research Laboratory (VDRL) test for Syphillis
Posted by: Azhar Hamdan (0503269C)

Introduction
Syphilis is a disease caused by the spirochaete organism Treponema pallidum. There are two types of serological tests that are useful for the diagnosis of primary, secondary and tertiary stages of syphilis.

Non-treponemal tests: Such as the Venereal Disease Research Laboratory (VDRL) test and the Rapid Plasma Reagin (RPR) test, which may be used as screening tests.

Treponemal tests: Such as the (TPPA) test and the LIA-Sphilis (Line Immuno Assay) IgM and LIA-Syphilis IgG tests. The LIA-Syphilis test is to be used as a confirmatory test depending on clinical situations.

The most common test requested would be the Venereal Disease Research Laboratory (VDRL) test (which is a non-treponemal test) and when this test turns out to be positive, we would then proceed with Treponema Pallidum Particle Agglutination (TPPA) test (which is a treponemal test).

VDRL is a non-treponemal test. Unlike TPPA (a treponemal test) which is based on the agglutination of gel particles sensitized with T. pallidum antigen by the patient’s antibodies, VDRL measures anti-lipid IgG and IgM antibodies called reagin directed agains substance in the mitochondrial membrane which are formed by the host in response to lipid from the treponemal surface.

Most of you would be quite familiar with TPPA, which is very similar to the Treponema Pallidum Haemagglutination (TPHA) tests we have done in school thus I would be focusing on the VDRL test only

VDRL Test using a Patient’s Serum Sample

Principle
The Venereal Disease Research Laboratory (VDRL) test is a slide microflocculation test for syphilis. It measures anti-lipid IgG and IgM antibodies called reagin directed against substances in the mitochondrial membrane which are formed by the host in response to lipid from the treponemal surface. The antigen which is suspended in buffered saline is composed of cardiolipin, cholesterol and lecithin and forms clumps when combined with lipoidal antibodies in serum from syphilitic patients.

If the serum is reactive, flocculation occurs due to the antigen reacting with the antibody. These complexes can be seen under the microscope. A reaction reported as weakly reactive shows small clumps, while if there are no clumps seen this is reported as nonreactive.

The antilipoidal antibodies are antibodies that are not only produced as a consequence of syphilis and other treponemal diseases, but also may be produced in response to non-treponemal diseases of an acute and chronic nature in which tissue damage occurs. Without some other evidence for the diagnosis of syphilis, a reactive non-treponemal test does not confirm T. pallidum infection.

Specimen Requirements
Heat serum obtained from centrifugation of clotted blood (5ml) at 56°C in a water bath for 30 minutes before testing. Cool down to room temperature. Examine the specimen after it is removed from the water bath. Re-centrifuge if debris is seen. Test sera within four hours after original heating. If there is a delay in testing reheat the sera at 56°C for 10 minutes. Refrigerate the sera at 2 to 8°C if there is a delay in doing the test. Avoid freezing and thawing of specimen repeatedly.

Equipment

Light microscope
Rotating machine adjustible to 180 rpm circumscribing a ¾ inch diameter circle.
Slide holders for 5x7.5 cm microscope slides.
Nondisposable calibrated needles without bevels, 18 gauge needle- for testing sera.
Slides 5x7.5 cm with ceramic rings approximately 14mm in diameter
2 ml syringe

Reagents
IMMUTREP® VDRL Antigen Ref OD011 from Omega Diagnostics Ltd is used. The antigen suspension is a colourless alcoholic solution containing 0.03% cardiolipin, 0.9% cholesterol and 0.2% lecithin. Antigens are stored in the dark at room temperature.

The VDRL antigen suspension has to be prepared following the manual included in the kit prior to use. This preparation is done daily as the antigen is not stable. Quality controls are tested before the antigen is used for patient samples.

Qualitative Test

Transfer 50 μL of serum into one ring of a ceramic-ringed slide.
Gently resuspend the VDRL antigen suspension.
Holding the VDRL antigen suspension dispensing needle and syringe in a vertical position, dispense several drops to clear the needle of air. Then add exactly 1 free-falling drop (17μL) of antigen suspension to each circle containing serum.
Place the slide on the mechanical rotator. Rotate the slide for 4 minutes at 180 ± 2 rpm
Immediately after rotating the slide, remove it from the rotator and read the test results.

Quantitative Test

Perform quantitative tests on all serum specimens that produce reactive, weakly reactive or “rough” non-reactive results in the qualitative VDRL test to their end-point titres.
Dilute Reactive sample from 1:1 to 1:32 (e.g., use 6 circles, number 1 to 6 for dilutions 1:1, 1:2, 1:4, 1:8, 1:16 and 1:32 respectively) and dilute Weakly reactive and rough Nonreactive sample from 1:1 to 1:4 by 2-fold dilution.
Add 50 μL of 0.9% saline in each of the circles except number 1 circle (1:1 dilution).
Transfer 50μL of serum in circle 1 and (to?) 2
Mix the saline and the serum in circle 2 by drawing the mixture up and down 7 to 8 times, transfer 50 μL diluted serum from circle 2 to circle 3 and mix thoroughly.
Repeat step 4 until the last circle, mix thoroughly and discard the last 50μL mixture from the last circle.
Gently resuspend the antigen suspension.
Following the way described in “Qualitative test” add 1 free-falling drop of antigen suspension to each circle.
Rotate the slide for 4 minutes at 180 ± 2 rpm and read the test.
If the highest dilution (1:32) is reactive, repeat the sample by higher dilution until the end-point.

Reading of test results

The results are read microscopically using 10X occular and a 10X objective.

Qualitative tests are read as follows:

Qualitative Method Report
Medium or large clumps Reactive (R)
Small clumps Weakly reactive (W)
No clumping or very slight roughness Non-reactive (NR)

Quantitative tests: The end-point titre is the last dilution that produces a reactive result.

Interpretation of Test Results
The VDRL test is an aid in the diagnosis of syphilis. A reactive VDRL may be unrelated to T. pallidum infection. The predictive value of a reactive VDRL in the serological diagnosis of syphilis is increasd when combined with a reactive treponemal test.

A reactive VDRL test may indicate past or present infection with a pathogenic treponeme. False-positive reactions can result from laboratory error as well as from serum antibodies that are unrelated to syphilis infection (1-2% of normal population, transiently post-immunization or febrile disease or during pregnancy)

A non-reactive VDRL test and no clinical evidence of syphilis may indicate no current infection or an effectively treated infection. A non-reactive VDRL test with clinical evidence of syphilis as a result of the prozone reaction, and in some cases of late syphilis, a fourfold rise in titre on a repeat specimen may indicate an infection, a reinfection, or a treatment failure; a fourfold derease in titre in early syphilis usually indicates adequate syphilis therapy.

All reactive qualitative VDRL tests should be diluted to an endpoint, and the endpoint titre should be reported. Unusually high VDRL titre can be seen with concurrent HIV-1 infection. Unusually high false-positive titres may be seen in some patients with lymhomas.

Lab Techniques

2007-10-28T08:59:00.000-07:00

Trichloroacetic acid(TCA) is one of the many organic solvents used in protein precipitation and purification. It works by disrupting the protein conformation thus exposing more hydrophobic areas, which reduces solubility of protein. In this way, the proteins will be precipitated.

We are using TCA to extract secretory proteins from our bacteria (S.maltophilia) and these are the steps involved.

Day 1 (Streaking)
1. Streak the strain on a LB Agar plate
2. Incubate plate for 24 hours at 37°C

Day 2 (1st Inoculation)
1. Inoculate an isolated colony into 20ml of LB broth
2. Incubate tube for 24 hours at 28°C
(Remark: The tube is incubated at 28°C instead of 37°C as most of the strains grow better at about 30°C. We'll like to the bacteria to proliferate so there will be enough cells for 2nd inoculation and for extraction)

Day 3 (2nd Inoculation)
1. Pellet cells at 3000xg for 20 minutes at 10°C
2. Decant the supernatant and resuspend the cells in 10mL of PBS

3. Spin down the tubes at 3000xg for 20 minutes at 10°C

4. Repeat steps 2 and 3.

5. Decant the supernatant and resuspend the cells in 20mL of PBS

6. Take OD600 reading in triplicates with 10X dilution
7. Calculate the volume withdrawal equivalent to 5.0X107 cells
8. Inoculate the calculated volume each into 4 tubes of 20ml LB broth
9. Incubate tubes at 37°C for 16 hours

Day 4 (TCA Precipitation)
1. Pellet cells at 3000xg for 20 minutes at 10°C
2. Suck up the supernatant (which contains the secretory proteins) with a syringe
3. Filter 18 ml of supernatant through 0.45um filter to remove contaminants (e.g. cell debris) into teflon tubes.
4. Add equal volume of chilled TCA into each tubes
5. Incubate the tubes -20°C freezer for an hour
6. Invert tubes every 10 minutes interval
7. Spin tubes at 16 000xg, 4°C for an hour

8. Decant the supernatant (beware of dislogded protein pellet)

9. Wash the protein pellets with 250ml of pre-chilled acetone
10. Transfer all protein pellets into a 1.5ml mircofuge

11. Centrifuge at 16 000xg at 4°C for an hour

12. Aspirate supernatant and air dry

13. Resuspend pellet with appropriate amount of rehydration buffer

Acetone is used to wash the cell pellet as TCA is also an organic solvent, like dissolves like so the TCA can be removed. Also,chilled acetone is preferred as low temperature allows the protein to stay in ppt form. When room temp acetone is used, protein pellet turns sticky and thus stick onto pipette tip and tube leading to loss of protein sample. Furthermore, the addition of organic solvent causes heat evolution, care should be taken to keep the temperature low.

Afterwhich, we'll do a protein quantitation to determine the concentration and run a 2D electrophoresis (refer to my first entry), followed by analysis of spots and excision of chosen spots(refer to Ming boon's entry). Finally, to run these peptides in MALDI TOF/TOF (refer to Shahirah's entry) to identfy these peptides. Scroll down if you want to read more about the principles of using these equipments.

That's all! It's the last week that we needa blog! YEAH. Hope you understand and learn something from my posting. See you people in school!

Tang Jiaxin (TG01)

Lab Techniques

2007-10-21T05:53:00.000-07:00

Hi guys...today i'll be talking about the washing procedures that Zahirah and I always have to do when there is insufficient sequencing plates and Axymats for our experiments. Hee... Feel free to ask any questions ya...

Title: Cleaning of sequencing plates procedure

Flood labeling on the sequencing plates with DMSO and wiping the sequencing plates dry and clean with tissue papers.
Rinse sequencing plates with tap H₂O.
Add decon 90into sonicator.
Place all sequencing plates into sonicator. Ensure they are totally submerged by using Styrofoam to press them down and the close the lid of the sonicator. Then add distilled H₂0 into the sonicator, filling it ¾ full.
Turn on the sonicator for 30min. (It will induce electrical impulses for washing of sequencing plates.)
After washing for 30min, turn on the tap of sonicator to allow the washing solution to be drained out into an empty pail and later been disposed into the sink.
Add decon 90 into sonicator again.
Rinse sequencing plates with tap H₂0 again.
Place sequencing plates into sonicator and add distilled H₂0,filling it ¾ full again. Place Styrofoam to press down the sequencing plates to ensure that they are fully submerged into the washing solution.
Turn on sonicator for 15 min.
After 15 min, turn on the tap of the sonicator to drain off the washing solution.
Rinse sequencing plates with tap H₂O again.
Take a beaker to fill up with milliQ H₂O.
Rinse the sequencing plates in milliQ H₂O for 4-5times.
Dry them using tissue papers.
Place them in the oven(65^oC) for 1 hour.
Wrap them with aluminium foil and sent them for autoclave.

Title: Cleaning of Axymats procedure

Prepare 0.5% to 1% sodium hypochlorite (bleach) solution. In this case, 1% bleach is prepared using M₁V₁=M₂V₂.

5.25% of bleach (stock conc) x V₁= 1% of bleach x 1500ml (total volume of bleach solution required)

Thus, V₁= 286ml (approx. 300ml)

Hence, add 300ml of 5.25% bleach into 1200ml of milliQ H₂0 and pour the solution into the sonicator. Turn on sonicator for 15 min after placing the Axymats into the prepared 1% bleach solution.

Rinse Axymats into 70% isopropanol and allow them to dry on tissue paper.
Place them in the oven for 30 min.
Wrap them with aluminium foil and sent for autoclave.

Michelle (0503808h)
TG02

SIP/MP sharing week 16

2007-10-12T07:02:00.000-07:00

Hey there! Yup it's my turn once again. Time really flies. It's week 16!!!

In this entry, i will be sharing with you my SIP/MP experience. As mentioned by both Jiaxin and Ming Boon, we have been assigned to be 'supervisors' to the DRP students for about a month. Ming Boon and myself have been tasked to supervise the students doing the protease project (refer to Ming Boon's post on 30 Sep). However, the students from the other project will also be supervised by us should both Johanna and Jiaxin be busy. As much as it was interesting, I must say I was thrown off guard even though I was fully aware of the fact that they had never worked in this conditions before. Frankly, I think I know how the lecturers feel now :)

I'd like to share with you guys an incident that happened recently. The group working on the extraction of periplasmic proteins using chlorofrom shock (refer to Jiaxin's post on 17 Sep) were supposed to run their sample on a 1-D gel (think MBio, Western Blot). They had prepared their samples as per protocol (or so i thought). After running their samples on the gel, it strucked them that they did not denature their samples.

For the benefit of everyone, I shall sidetrack and explain the flow for running a pre-casted 1-D gel.

Basically, you will take out a certain volume of the samples into a new microcentrifuge/eppendorf tube. In this case, it was 10ul. After which, you will add an equal volume of a mix of sample buffer (I'm not sure what's in it but it contains Bromophenol Blue and SDS [gives the proteins an overall negative charge]) and B-mercaptoethanol. Any idea what's B-mercaptoethanol is for? It is a strong reducing agent that breaks the disulfide bonds found in proteins. (This step is done under the fume hood as B-mercaptoethanol gives off a very pungent smell.)

This image was taken from http://tonga.usip.edu/gmoyna/biochem341/bme.gif

Once you have done all that, you will centrifuge the tubes for 4-6 secs to collect all the liquid down. Then you will heat the samples up at 95°C for 5 mins to denature the proteins. Following this, the tubes will be centrifuged briefly again to collect all the liquid down. The samples are now ready to be loaded onto the gel.

The gel is removed from its packaging (the gel is pre-casted) and placed in the gel tank. All the samples are loaded in the wells, except for the first and the last wells where the protein ladder/marker will be added. You will then top up the buffer in the gel tank if needed and the gel is noe ready for electrophoresis. Yup so that's the steps done when running a 1-D gel. For those who are interested to know what happens after that, leave me a comment aite?

Back to the actual story. As I'd said earlier, they did not denature their samples . This means that the proteins will still be in their native conformation and thus affect their rate of migration in the gel, which in a way is pointless as the main principle of 1-D gel is to separate the linearized proteins based on their mass.

This will be one incident that I will not forget.

That wil be all for this week's post. To all, Selamat Hari Raya and hope you will have a good weekend. Four more weeks to go!!!! Take care and see you guys during the next campus discussion!

Posted by Shahirah Bibi Tg01, 0503174E

MMIC

2007-10-05T05:08:00.000-07:00

Procedure: Biochemical Testing for Neisseria gonorrhoeae

Posted by: Azhar Hamdan (0503269C)

For the moment, I am temporarily attached to the Serology Lab for 2 weeks, with my first week being in the STD Lab. Yeap, thats rite Sexually Transmitted Diseases. I know what you are thinking, "Eeeks HIV!" However, on the contrary , the lab only tests for 2 organisms, Neisseria gonorrhoeae (causes Gonorrhoeae) and Treponema Pallidum (Syphilis). So no HIV here :).
For this post, I am going to try adding as much pictures as possible to keep you guys interested alrite?

Anyways my post will focus more on N. gonorrhoeae, a.k.a Gonococci or GC.

The common types of specimens received by the lab are swabs from:

Endocervix
Urethra
Anorectal
Oropharynx
Conjunctiva
Bartholin gland
Skin lesions
Joint fluids

And plain blood specimens.

An interesting point to note is that more than half of the specimens come from Sex Workers that regularly go for checkups to retain their license. So people, always stay protected!

Anyways, upon receiving the specimens, the lab staff would process the specimens and allocate lab numbers. They would then inoculate & streak the swabs onto GC -Lect Agar, a selective agar that promotes the growth of GC that contains antibiotics that would suppress the growth of other organisms such as gram-positive bacteria, including vancomycin-resistant Staphylococcus epidermidis, gram-negative species like Proteus and Capnocytophaga, as well as fungi including Candida albicans. The plates would then be incubated at 35-37°C for 24 hours.

All the above is done by the lab staff.

After incubation, the plates are read. Suspected GC colonies undergo a Gram-stain & 4 biochemical tests (these were practised by us students):

Oxidase test to test for the production of oxidase
Superoxol test (using 30% Hydrogen Peroxide [H₂O₂]), similar to the Catalase test (using 3% H₂O₂), to test for the production of catalase
Rapid Carbohydrate Degradation Test (Sugar Test), to test for the ability to degrade sugars)
β-Lactamase test, to test for the production of penicillinase which confers resistance to penicillin

Only the first three are confirmatory tests. The last test is a susceptibility test to check for resistance to penicillin.

The oxidase test and superoxol test (similar to catalase) are some common tests that we have learnt in Basic Microbiology so I shall not go into details, however, questions are welcomed.

I chose the Rapid Carbohydrate Degradation Test (Sugar Test) to elaborate on, as it is so different from all the other tests that checks for the utilisation of sugar (e.g. TSI & KIA). Specimens are tested with the sugars individually. The sugars used in this test are 10% solutions of Glucose (G), Maltose (M), Lactose (L) and Sucrose (S)

Rapid Carbohydrate Degradation Test (Sugar Test)

Principle
This is a non-growth method which depends upon pre-formed enzymes. A heavy inoculum of organism degrades the sugar solutions containing the phenol red indicator. A colour change occurs in 2 to 4 hours.

Procedures

A heavy suspension of the organism is made (using a platinum inoculating loop) in a test tube of 1.0mL Buffered Salt Solution (BSS), to contain approximately 10⁹organisms per mL. Controls were also done together with the patient specimen. These controls are N. gonorrhoeae CDC 117 strain (117), N. lactamica (LC), Branhamella catarrhalis (BC) & Staphylococcus epidermidis (S)The BSS is red in colour due to the phenol red indicator.
This suspension is mixed well with a Pasteur pipette (Two Pasteur pipettes are available, use the bigger pipette [top] for this step).
Using the bigger Pasteur pipette, two drops of the suspension are delivered into each of the five wells of a microtitre plate which are labeled horizontally across the plate as Control (C), Glucose (G), Maltose (M), Lactose (L) & Sucrose (S).[The group of wells on the left side were from a previous practice, microtitre plates are shared to avoid wastage]
Using the smaller Pasteur pipette, one drop of each of the 10% carbohydrate solutions (G, M, L and S) is added carefully to each of the appropriate wells, making sure there is no splashing.
Cover the microtitre plate and mix well using the micro-shaker.
Incubate at 35-37°C for 2-4hrs
After incubation, examine plate for colour change. A red to yellow colour change indicates a positive reaction. Control well should remain an orange/red colour which indicates a negative reaction.
Results for the above test (both left & right specimens):
- First row, patient specimen: positive for G.(N. gonorrhoeae)
- Second row, 117: positive for G.
- Third row, LC: positive for G, M & L.
- Fourth row, S: positive for G,M & S
- Fifth row, BC: all negative

All the results tally with the expected results.

Pictures were taken with permission from the supervisor. Thanks Andre for being my model. Haha.

So thats all.

5 more weeks...Ahhhh!!!

Lab Techniques

2007-09-30T00:38:00.000-07:00

Hi everyone,

Like Jiaxin and Johanna, I am also the ‘supervisor’ of the DRP students. Shahirah and me are taking the group that will be doing a project that involves proteases secreted by Stenotrophomonas maltophilia at 37^oC. We would have to teach them how to prepare the necessary media, teach them the things that they do not know (eg. how to streak in the proper way) and guide them through the experiment so that it is successful.

This is a very simple project and the purpose is to find out which are the strains that produce a lot of proteases. Strains that produce a lot of proteases might be more virulent as the proteases might be involved in the degradation of the host proteins involved in the immunity eg. Immunoglobulins (Ig).

Principle:

After growing the colonies, the colonies will be transferred onto milk agar. If the strain produces proteases, the proteases will digest the milk proteins and a clearing that looks like a halo around the colony will be observed.

Procedure:

Day 1 - Streaking of environmental strains

Streak the strains onto LB agar (Plate is divided into 2 as there are only 2 strains and only abit of the colony is needed)
Incubate the plate at 37^oC for 48 hours (As environmental strains do not grow very well at 37^oC, more time is required for them to grow)

Day 2 - Streaking of clinical strains, negative and positive control

Streak Escherichia coli (negative) and Pseudomonas fluorescens (positive) onto a LB agar each
Streak the strains onto LB agar (Plate is divided into 4)
Incubate at 37^oC for 24 hours

Day 3 - Transferring of colonies to milk agar

Divide and label the milk agar appropriately
Use a sterilised toothpick to touch the colony and then dab on the milk agar gently
Incubate the plates at 37^oC
Observe for halos (This will take about 3 days of incubation)

That’s all! Thanks=]

Chua Ming Boon

Tg01

0503197F

Florescence in situ hybridization

2007-09-24T17:52:00.000-07:00

Florescence in situ hybridization (FISH), is a technique involving the separation of double stranded DNA by heat and hybridization of the target DNA sequence to the respective probes with a complementary sequence deirectly-labeled with a florochrome. The metaphases to which the probes anneal to are visualized as florescent signals of specific wavelengths under fluorescence microscopy. The number of signals observed in the metaphase spreads/interphase cells would indicate the copy number of the gene/gene loci concerned. This technique allows the identification of microdeleted/duplicated genes, aneuploidy screening (detection of extra chromosomes of chromosomes 13, 18, 21 X and Y), translocated genes in haematological malignancies and gene amplification from cases with cancer of the breast.

I would be explaining on identifying deleted/duplicated genes and loci that are related with microdeletion syndromes in congential disorders such as Prader-Willi/Angelman, Williams and Di-George using FISH techniques.

Samples – cultures of PHA(mitogen)-stimulated lymphocytes cells, amniotic fluids and chorionic villi.

Slide Preparation

1. Etch out the area on the underside of the glass slide with a diamond pen that contains the highest number of metaphase spreads. Slides can be kept in an airtight container at -20°C or 4°C if they are not used immediately.

2. Dehydrate the slide through an ethanol series (70%, 85% and 100% respectively) for 1 minutes each at room temperature. Blow-dry the slide with a hair dryer.

Probe Preparation and hybridization
The following steps must be carried out in reduced lighting as the probes are light-sensitive.

1. Remove the probe vial from the freezer and thaw at room temperature. Centrifuge the vial.

2. Add probe mixture onto the etched area of the slide and cover with a circular glass coverslip.

3. Seal the edges with rubber cement and place the slide into a light proof box.

4. Switch on the 'HYBrite' system and set the desired program (denaturation and hybridization steps).

5. Moisten the toweling paper in the troughs with water. Place the slide onto the hybridization surface, coverslip facing upwards.

6. Ensure that the target area is in contact with the hybridization surfaces and also that all the remaining slide positions are filled with blank slides to even out the temperature.

7. Close the 'HYBrite' lid and commence the program.

Post Hybridization Wash
Carry out the following steps in reduced lighting.

1. Switch on water bath. Set the temperature to 75° C. Prepare two coplin jars for Wash A and Wash B or two plastic slide chambers if only 1 or 2 slides are required for washing.

2. Place the coplin jar/slide chamber containing Wash A in to the water bath when it is just switched on.

3. Leave the coplin jar/slide chamber containing Wash B at room temperature.

4. When the water bath reaches its set temperature, remove the slide from the 'HYBrite' system. Peel off the rubber cement from the coverslip with a pair of forceps. Remove the coverslip.

5. Place the slide in the Wash A and agitate the slide by jiggling briefly for about 3 seconds. Wash for exactly 2 minutes.

6. Remove the slide and transfer to Wash B. Agitate the slide by jiggling briefly for about 3 minutes. Wash for 1 minute.

7. Remove the slide and air or blow dry completely in the dark at room temperature. Apply 10m l of DAPI counterstain (Vectashield mounting medium) and mount a glass coverslip over the hybridize area. Seal with nail vanish.

Image of Digeorge Syndrome

Adapted from :
http://www.pathology.washington.edu/galleries/Cytogallery/main.php?file=intro

Image of HYBrite system

Adapted from:

http://www.ebiotrade.com/buyf/productsf/vysis/img189_1.jpg

Reference:

American College of Medical Genetics Laboratory (1999). Standard guidelines for clinical genetics laboratories, 2nd Ed. Bethesda, MD: ACMG.

By:

Yvonne Lau

0503149G

TG01

Lab Techniques

2007-09-17T08:38:00.000-07:00

HELLO! =D

If you're wondering what we the in-house students are doing for SIP. NO NO.. not preparing reagents and stock checks! For our group, our assigned task is to be the "supervisors" of the DRP (Differential Research Programs) students!

Johanna and I will be taking one of two groups who are under Dr. Quek's project. This project involves extraction of periplasmic proteins using chloroform (CHCl₃) shock, of an isolate of Stenotrophomonas malitophilia which is incubated at two different temperature 28°C (environmental) and 37°C(human body temp.). The differences in protein expression will be analyzed and compared. Experiment will be repeated on other isolates too. This is also the continuation of what Jo and I have been doing for our last DRP.

Outline of Workflow/Protocol

Day 1 (Streaking)

1. Streak the different isolates on a well divided LB Agar plate
2. Incuabte plates for 24 hours at 37°C

Day 2 (Inoculation)

1. All colonies of each isolates is scrapped off and mix in 5mL PBS
2. Cell density (Opitcal Density 600) taken in triplicates to determine number of cell present
3. Volume of inoculum equivalent to 5.0X107 is inoculated into 2 tubes
4. One tube incubate at 28°C and the other at 37°C for 16 hours 250 rpm

Day 3 (Extraction)

1. After 16 hours, the tubes are spin down at 3000xg for 20 minutes at 10°C

2. Decant the supernatant and resuspend the cells in 10mL of PBS

3. Spin down the tubes at 3000xg for 20 minutes at 10°C

4. Repeat steps 2 and 3.

5. Decant the supernatant and resuspend the cells in 10mL of PBS

6. Take OD600 reading in triplicates with 10X dilution

7. Calculate the volume withdrawal equivalent to 1010 number of cells

8. Pipette the calculated volume into 1 microfuge tube

9. Spin down cells at 3000xg for 20 minutes at 10°C

10. Decant supernatant

11. Resuspend pellet in remaining medium by brief vortexing

12. Add 20mL of chloroform to each tube

13. Incubate at room temperature for 15 mintues

14. Add 200uL of 0.1M Tris-HCl to tubes to stop reaction

15. Spin tubes at 6000xg for 20 minutes at 10°C

16. Collect the supernatant to a new microfuge tube

17. Spin the supernatant tubes at 14000xg for 10 minutes at 10°C

- This is to ensure that only the periplasmic proteins are present in the supernatant as cell debris will be pellet down

18. The supernatant is pipetted out into a new microfuge tube

Example of how to calculate 5.0X107 worth of cells

Average OD600 Reading = 1.70

Desired number of cells = 5.0X107

1.00 OD = 1.4X107 cfu/mL

Volume of inoculum = (Desired no. of cell) / (Average OD600 X 1.4X107) X1000

= (5.0X107) / (1.70 X 1.4x107)

= 21.0uL

After extraction, protein concentration will be determined by Bradford assay. The samples will be run on 1-D gel (separation by MW) and stained with either Coomassive Blue or Silver stain. Protein bands will be analyse and compare to identify the difference in protein expression of an isolate incubated at 28°C and 37°C.

Post Stained 1D Gel
Adapted from: http://www.raytest.de/bio_imaging/applications/gel_documentation/applications_geldoc1.jpg

As this is a project that lasts for only 3 weeks, the job load is lesser and simpler.

That's all. Have great day!

Tang Jiaxin TGo1

0503257H

Lab Technique

2007-09-09T06:17:00.000-07:00

Hiya...I shall talk about purification of primers and followed by DNA sequencing after finding the optimization temperature,a lab technique that I have done after PCR. The purpose of purification is to ensure that the unwanted unbound stuff like unbound primers or excess buffer will not be present and thus will not affect the results after DNA sequencing is done. Feel free to ask any questions ya..

Purification of PCR Products:

Materials

Shrimp Alkaline phosphotase (SAP)

Exonuclease 1 (Exo 1)

Method:

1) Prepare a master-mix containing both the enzymes for purification. (Refer to table below for purification reaction mixture set-up.)

Purification reaction mixture set-up

Components Required:	Volume Required/ul:	Master-mix Required/ul:
SAP	1.0	80
Exo 1	0.5	40
	1.5	120

2) Pipette 15ul into 8 eppendof tubes. Using a multi-channel, pipette 1.5ul of the master-mix into the wells of the PCR plates.

3) Place the PCR plates into the machine.

DNA Sequencing (Cycle sequencing):

Materials:

Sequencing dye

Sequencing buffer

Forward primer

Reverse primer

Autoclaved water

Method:

1) Prepare a master-mix containing all the components for cycle sequencing. (Refer to table below for cycle sequencing reaction mixture set up.)

Cycle sequencing reaction mixture set up

Components required:	Volume Required/ul:	Master-mix Required/ul:
Sequencing dye	1.0	80.0
Sequencing buffer	0.5	40.0
Forward primer OR reverse primer	0.4	32.0
Autoclaved water	0.6	48.0
	2.5	200

2) Pipette 2.5ul of the required reaction mixture into a new PCR plate.

3) Pipette 2.5ul of the correct purified PCR products into the new PCR plate, which contains the reaction mixture. Ensure it is mix well.

4) Spin down the new PCR plates.

5) Place the new PCR plates into the machine.

DNA Sequencing (After cycle sequencing):

Materials:

3M of Sodium acetate

125mM of EDTA

100% Ethanol

70% Ethanol

Formamide

Method:

1) Spin down the PCR products in the PCR plates.

2) Prepare a master-mix of the stock solutions (200ul of 3M sodium acetate and 200ul of 125mM of EDTA).

3) Pipette 2ul of master-mix of the stock solutions into each well of the PCR plate, using a multi-channel.

4) Dispense 25ul of 100% ethanol into each well of the PCR plat, using a multi-channel.

5) Pipette everything from the PCR plates and transfer them into sequencing plates, using a multi-channel.

6) Wrap the sequencing plates with aluminum foil and incubate them at room temperature for 15 minutes. Dispose the PCR plates.

7) Centrifuge the sequencing plates at 3000G at 4^oC for 25 minutes.

8) Invert the sequencing plates using 3 pieces of tissue paper and pulse them at 200G at 4⁰C for 20 seconds.

9) Dispense 70% ethanol into each well of the sequencing plates.

10) Centrifuge the sequencing plates at 1700G at 4^oCfor 15 minutes.

11) Invert the sequencing plates using 4 pieces of tissue paper and pulse them at 200G at 4^oC for 20 seconds.

12) Repeat step 11, using the other sides of the tissue paper.

13) Place the sequencing plates into a vacuum dryer, not more than 7 minutes.

14) Pipette 20ul of formamide into each well of the sequencing plate/

15) Cover the sequencing plates with rubber mats and place the sequencing plates in the septa. Place them into the sequencer machine.

Michelle TG02 0503808H

Lab Techniques

2007-09-02T03:12:00.000-07:00

CyDye Fluor in DIGE

Hi guys! Hope all of you are doing well in the 10th week of your attachment, 10 more weeks to go so hang in there all right. For those who are enjoying your work, appreciate every week cause all good things must come to an end :p

For this blog entry, I will be sharing with you guys about CyDye used in DIGE. DIGE stands for DIfferential Gel Electrophoresis. It is a technique used to detect and identify proteins involving 2D-gel electrophoresis (scroll down to Jiaxin’s entry for more info). It allows up to three different protein samples to be separated on the same gel. This ability is termed multiplexing. Each protein sample is labeled with one type of dye i.e. CyDye in this case.

Now what is CyDye then? Basically it is a cyanine, fluorescent dye used to label the proteins. There are different forms of CyDye depending on the sample you are dealing with. For example there is a set of CyDye to label scarce samples. Since the sample is limited, a lot of dye is added and bind to the proteins at certain amino acid residues such that they become saturated with the dye. The saturation will help in the detection of the presence of the proteins after 2D- gel electrophoresis. In general, CyDye is excited lasers at specific wavelengths and emits a signal of a narrow wavelength range. There are three CyDyes used in most experiments; Cy2, Cy3 and Cy5. Cy2 is excited by a laser at a wavelength of 488nm whereas Cy3 is excited at 532nm and Cy5 at 635nm.

For my group’s experiments, we will most probably use one of the three dyes i.e. Cy3. We are only using one as we intending to test if this method of labeling is suitable for the bacterium that we are working on i.e. Stenotrophomonas maltophilia. At the moment we are trying out a protocol to extract only the exposed cell surface proteins of this gram-negative bacterium and we are using this dye to label the surface membrane proteins.

As mentioned earlier that there are various forms of cyanine dyes depending on the experiment. The Cy3 that we are using is a minimal dye. This dye has an ester group on it. It will bind only to the epsilon amino group of lysine found on proteins to form an amide linkage. Most proteins have a number of lysine residues on them thus these proteins will be labeled with the cyanine dye. It is termed a minimal dye as it is limiting in the reaction between the proteins and the dye. Only a small amount of the dye will be added to ensure that only 1-2 % of the available lysine is labeled with the dye. This is so as during identification of the proteins using mass spectrometry like MALDI (refer to my previous post), the proteins will be cut using trypsin. Trypsin cleaves at lysine. Thus, we use minimal labeling such that it will not affect the mass spectrometry results. Once the proteins are labeled, the sample will be subjected to gel electrophoresis. For my experiment, the sample will be run on 1D-gel where the separation of proteins is based solely on its molecular weight.

That will be all for my post. Take care and say hi to week 11!

Posted by: Shahirah Bibi
0503174E
TG01

Microbiology

2007-08-25T05:42:00.000-07:00

Topic: Mycology
Procedure: Fungal Culture
Posted by: Azhar Hamdan (0503269C)

In this post, I am focusing on the fungal culture procedure for clinical specimens.

Purpose

Fungal cultures on clinical specimens are done to detect the presence of:
• Yeast
• Yeast-like fungi
• Hyaline or dematiaceous filamentous fungi
• Aerobic actinomycetes

Common specimens received
• Cerebrospinal Fluid (CSF)
• Blood
• Vaginal/Cervical Swabs
• Skin
• Hair
• Nail

Media Used
• Sabouraud Dextrose Agar (SDA)
• Sabouraud Dextrose Agar with Chloramphenicol (SDC)
• Brain Heart Infusion Agar with Chloramphenicol and Gentamicin (BHICGA)
• Brain Heart Infusion Agar (BHIA)
• Mycobiotic agar with Actidione (AA)
Chloramphenicol, Gentamicin & Actidione are antibiotics. The purpose of adding antibiotics to media is to suppress normal flora and thus to allow for the multiplication and detection of the pathogenic organisms

Okay, maybe all the media looks the same, but just to point something out, different media have different corresponding coloured covers. This prevents accidental mix up.
(Left to right; SDA, SDC, BHICGA, BHIA & AA)

General Procedures
• The ideal temperature for the recovery of fungi is 30°C
• Cultures are kept for 2-4 weeks depending on the specimen and the suspected organism before cultures are reported as negative and then discarded
• Media is preferably prepared as a slant in a tube as opposed to being prepared in a Petri-dish

Reasons for preference of slant:
• Potentially less hazardous for staff to work on ( e.g. due to the vertical tubes, aerosols produced would less likely escape)
• Less prone to contamination and dehydration
• Easier to store in racks

Pictures were taken with permisson from the supervisor.

Thats all for this post. Say HELLO to week 10