BloodBank.MedMic.Haematology

Monday, December 10, 2007

Case 1 Posting 2

Subject: MMIC
Case: Case 1 - Khong Fay Seah
Complaints: Fever, chills, dysuria,
Diagnosis: Urinary Tract Infection

List of OrganismsTests Required to Identify Expected Test Results Treatment
Escherichia Coli
  1. Culture
  2. Gram stain and Microscopy
  3. Biochemical test test – TSI, IMViC (Indole, Methyl red, Voges-Proskauer, citrate), motility
  1. Lactose-fermenting (Yellow colonies in CLED); Flat, grey colonies on BAP
  2. Gram-negative bacilli
  3. Biochemical test
    TSI - Acid slant acid butt with gas but no H2S
    Indole – Positive (Bright red interface)
    Methy red – Positive (Red)
    Voges-Proskauer - Negative
    Citrate – Negative (Remains green)Motility – Motile (Turbidity out of line of inoculation stab)
Trimethoprim-sulfamethoxazole ampicillin
Klebsiella spp.
  1. Culture
  2. Gram stain and Microscopy
  3. Catalase test
  4. Biochemical test – IMViC (Indole, Methyl red, Voges-Proskauer, citrate), motility
  1. Lactose-fermenting (Yellow colonies in CLED); Mucoid, convex, white colonies on BAP
  2. Gram-negative bacilli
  3. Positive catalase test (Effervescence)
  4. 1) Biochemical test
    Indole – Negative (No colour change)
    Methy red – Negative (Yellow)
    Voges-Proskauer – Positive (Colour change)
    Citrate – Positive (Deep blue)Motility – Nonmotile (Growth along stab line)
Cephalosporins Aminoglycosides
Streptococcus spp.
  1. Culture
  2. Gram stain and Microscopy
  3. Catalase test
  1. Lactose-fermenting (Yellow colonies in CLED); Small, white colonies on BAP
  2. Haemolysis:

    • α-haemolytic:
      eg. Streptococcus pneumoniae
    • β-haemolytic:
      Streptococcus group A, B etc
    • Non-haemolytic:
      E.g. Enterococcus spp.(Greyer appearance)
  3. Gram-positive cocci
  4. Negative catalase test
Penicillin Eryhromycin Aminoglycoside
Staphylococcus spp. Eg. S saprophyticus, Staphylococcus aureus
  1. Culture
  2. Gram stain and Microscopy
  3. Catalase test
  1. Lactose-fermenting (Yellow colonies in CLED); Small, white colonies on BAP
    • Can be hemolytic positive or negative
  2. Gram-positive cocci
  3. Positive catalase test (effervescence)
Penicillins /with clavulanic acid
Cephalosporins Vancomycin
Pseudomonas spp.
  1. Culture
  2. Gram stain and Microscopy
  3. Oxidase test
  4. Biochemical test - TSI
  1. Non-lactose-fermenting; Swarms on BAP; Spready colonies on CLED; Pungent smell
  2. Gram-positive bacilli
  3. Positive oxidase test
  4. Biochemical testTSI - Alkaline slant acidic butt
Penicillin Aminoglycoside
Proteus spp.
  1. Culture
  2. Gram stain and Microscopy
  3. Oxidase test
    Biochemical test - TSI, Urease
  1. Non-lactose-fermenting; Swarms on BAP; Glossy colonies; Pungent smell
  2. Gram-negative bacilli
  3. Negative oxidase test
  4. 1) Biochemical test
    TSI - Alkaline slant acidic butt with H2SUrease – Positive (Purple/pink)
Aminoglycosides
Trimethoprim-sulfamethoxazole
Ampicillin Cephalosporins
Candida spp.Eg. Candida albicans
  1. Culture
  2. Gram stain and Microscopy
  1. Non-lactose-fermenting; Small, flat, white colonies on BAP and CLED; Slightly spready, not glossy
  2. Fungus; pseudohyphae
-
Enterococcus faecalis
  1. Culture
  2. Gram stain and Microscopy
  3. Catalase test
  4. Biochemical test – Motility, Indole
  1. Large white colonies; alpha-, beta-, nonhemolytic colonies
  2. Gram-positive cocci
  3. Negative catalase test
  4. Biochemical test Motility – Nonmotile (Growth along stab line)Indole – Negative (No colour change)

Penicillin Vancomycin Gentamicin

Morganella
  1. Culture
  2. Gram stain and Microscopy
  1. Large, gray, moist colonies (beta haemolytic)
  2. Gram-negative bacili
Aminoglycosides Trimethoprim-sulfamethoxazole
Serratia
  1. Culture
  2. Gram stain and Microscopy
  3. Catalase test
  4. Biochemical test – IMViC (Indole, Methyl red, Voges-Proskauer, citrate), motility
  1. Lactose-fermenting but may produce negative results; Red-pigmented colonies
  2. Gram-negative bacilli
  3. Positive catalase test (effervescence)
  4. 1) Biochemical test
    Indole – Negative
    Methy red – Variable
    Voges-Proskauer - Positive
    Citrate – PositiveMotility – Motile (Turbidity out of line of inoculation stab)
Cephalosporins Aminoglycosides


Photos of Various Microorganism on Agar Plates:
E.Coli
Klebsiella spp.
Pseudomonas & Klebsiella spp.
Proteus spp.
Candida spp.


Photos are taken by our bacteriology group to "xinjiapo"-GH (Andre,Azhar,Valerie,Boonching)

References:
1. MedlinePlus. (2007) Urinary Tract Infection. Retrieved December 02, 2007, from http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm
http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm > >urinary tract infection
2. MedicineNet, Inc. (2007) Definition of Dysuria. Retrieved December 02, 2007, from http://www.medterms.com/script/main/art.asp?articlekey=3163
http://www.medterms.com/script/main/hp.asp > dysuria
3. Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

Boon Ching
TG01

MMIC CASE 6


Particulars of Patient
Name: Wong Fei Fei
Sex: Female
Age: 37
Complaints: Fever, pain during urination, vaginal discharge

List of possible organismTest requiredExpected resultstreatment

Bacteria

Pseudomonas aeruginosa


1) Gram Stain

2) Biochmical Tests:
- Catalase test
- Oxidase test
- TSI agar test


3) Aerobic culture on plates like MacConkey agar plate, blood agar plate or differential media


4) Antibiotic Susceptibility (aminoglycoside + penicillin)


1) Shows gram negative rod

2) Catalase and oxidase positive
TSI medium no colour change (K/K/g-/H2S-)

3) Colourless colonies on MacConkey plateColonies grown on blood agar.






Antibiotics that have activity against P. aeruginosa include:
- aminoglycosides
- quinolones (ciprofloxacin)
- cephalosporins (ceftazidime and cefoperazone)

Escherichia coli

1) Gram Stain


2) Biochemical Tests:
- TSI agar test
- Methyl red test
- VP reaction test
- Citrate test


3) Aerobic culture on MacConkey agar or EMB agar (or both), blood agar


4) Serology is done using the SSS-Coagglutination test


5) Antibiotic Susceptibility (fluoroquinolone)


1) Gram negative rods


2) • Grows on TSI slant with a (A/A/g+/H2S-) profile
• Indole (+)
• Methyl red (+)
• VP (-)
• Citrate (-)


3) On MacConkey agar, deep red colonies are produced.


Growth on EMB agar would show black colonies with greenish-black metallic sheen.


Hemolysis on blood agar


4) Serology detects O55:K5:H21


Antibiotics which may be used to treat E. coli infection:
- nitrofurantoin
- fluoroquinolones
- meropenem
- aminoglycosides
- ciprofloxacin

Enterococcus spp.

1) Gram Stain

2) Biochemical tests:
- Catalase test


3) Antibiotic Susceptibility (gentamicin + vancomycin)


1) Cocci in tetrad and clusters

2) (-) to weakly (+) catalase


Can treat with vancomycin and gentamicin






Proteus mirabilis

1) Gram stain

2) Biochemical tests:
- Indole test
- Methyl red test - VP test
- Catalase test
- Oxidase test
- Urease test


3) Aerobic culture on XLD plate, blood agar or differential media


4) Antibiotic Susceptibility (ampicillin)


1) Gram negative rod

2) Indole test (-)
Methyl red (+)
VP (-)
Catalase (+)
Oxidase (-)
Urease (+)


3) Colonies growns on the agar plates


Antibiotics which may be used to treat:

- ampicillin
- aminoglycoside
- TMP-SMZ
- fluoroquinolone
- cephalosporin


Chlamydia Trachomatis




1) McCoy cell culture


2) Fluorescent Antibody test/Enzyme-linked Immunoassay


3) DNA probe test


4)Antibiotic Susceptibility (Doxycycline or azithromycin)


1) Cytoplasmic inclusions observed by immunofluore-scence after few days of incubation in cell culture


2) Detect the presence of genus-specific lipopolysaccharide antigen


Can be cured with antibiotics like:


- Azithromycin
- Doxycycline
- Tetracycline

Neisseria Gonorrhoeae

1) Gram stain


2) Biochemical Test
- Oxidase test


3) Serology Antibiotic Susceptibility (Penicillin)


1) Gram negative, diplococcus


2) Oxidase (+) IgG and IgA antibodies against gonococcal pili, outer membrane proteins and LPS detected



Recommended treatment:

- cephalosporin
- Fluoroquinolone plus an antibiotic e.g. ceftriaxone, cefixime, ciprofloxacin

Klebsiella pneumoniae

1) Gram stain


2) Biochemical test:
- Citrate test
- VP test


3) Aerobic culture on blood agar, MacConkey agar and differential media


4) Antibiotic susceptilibilty (cephalosporin)


1) Gram negative, large and regular capsules


2) Citrate (+)
VP (+)


3) Large colonies and very mucoid






Can be cured with antibiotics like:


- cephalosporine
- aminoglycoside
- imipenem

Providencia species

1) Gram stain


2) Biochemical test:
- Urease test


3) Aerobic culture on blood agar and differential media


4) Antibiotic susceptilibilty (ceftazidime and cefepime)


1) Gram negative


2) Urease (-)


3) Circular, convex, smooth colonies






Recommended treatment with:

- ceftazidime
- cefepime
- aztreonam
- imipenem

Citrobacter

1) Gram stain


2) Aerobic culture on blood agar and differential media


3) Biochemical test:Citrate test



1) Gram negative


2)Circular, convex, smooth colonies


3) Citrate (+)




Antibiotics which may be used to treat:

- ampicillin
- cephalosporins
- fluoroquinolones

Viruses

Herpes simplex virus type 2 (HSV-2)

1) Virus isolation using inoculation of tissue culture


2) Serology


1) Viral DNA identified


2) Antibodies of HSV-2 detected






Several antiviral drugs are effective against such infection:
- Acyvlovir
- Valacyclovir
- Vidarabine

Parasites

Trichomonas vaginalis

1) Microscopic examination – vaginal discharge examined microscopically in a drop of saline


2) Anaerobic culture


1) Characteristic motile trichomonads observed in wet mount


2) Colonies growth on agar plates






Successful treatment requires destruction of trichomonads by medicine like:
- metronidazole (e.g. Flagy)
- tinidazole and ornidazole

Fungi

Candida albicans

1) Potassium hydroxide (KOH) preparation of the vaginal discharge


2) Culture on fungal or bacteriologic media at room temperature or 37oC


1) Hyphae and pseudo spores of Candida are visible under microscopic examination


2) Culture medium is incubated for several days, during which time colonies of yeast and/or bacteria develop










Usually treated with antimycotics—antifungal drugs e.g. topical clotrimazole, topical nystatin, fluconazole, and topical ketoconazole



Images of possible microorganisms:



Pseudomonas aeruginosa
on mac conkey plate (1)



P. mirabilis on an XLD agar plate (2)





C. trachomatis inclusion bodies (brown) in a McCoy cell culture (3)



trichomonas vaginalis wet mount (4)



Candida albicans (4)


K. pneumoniae on a MacConkey agar plate (5)



References:
1. http://www.austincc.edu/microbugz/html/ > macconkey_agar
2. http://en.wikipedia.org/wiki/Main_Page > Proteus_mirabilis
3. http://en.wikipedia.org/wiki/Main_Page > Chlamydia_trachomatis
4. http://www.google.com/ > image search > "microorganism name"
5. http://en.wikipedia.org/wiki/Main_Page > Klebsiella_pneumoniae



Doreen Ng (TG 01)

Case 3 2nd Post

Possible Micro-organismMicroscopyCultureBiochemical TestsAntibiotics Sensitivity TestingTreatment
Escherichia ColiGram negative bacilli

Mac Conkey: Lactose Fermenting(Pink)

Sheep Blood Agar: β-hemolytic

TSI: acidic slant/ acidic deep

Oxidase Test: Negative

IMViC: ++--

Urease Test: Negative

Sensitive to amoxillin, cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin, aminoglycosidesNo drug is normally needed. Amoxillin used when needed.
Proteus spp.Gram negative bacilli

Mac Conkey: Non-Lactose Fermenting(colourless)

Sheep Blood Agar: Spreading Growth

TSI: alkaline slant with black ppt

Oxidase Test: Negative

IMViC: ++-+*

Urease Test: Positive

Resistance to tetracycline, first generation cephalosporins and ampicillinsMost antibiotics can be used other then the 3 that it is resistant to.
Klebsiella spp.Gram negative bacilli

Mac Conkey: Lactose Fermenting(Red)

Growth in areobic and anareobic condition

TSI: alkaline slant/ acidic deep

Oxidase Test: Negative

IMViC: --++

Urease Test: Negative

Susceptible to aminoglycosides cephalosporin, gentamicin

Resistance to carbenicillin, ampicillin, quinolones, and increasingly to ceftazidime

Genamicin
SerratiaGram negative bacilli

Mac Conkey: Non-Lactose Fermenting(Colourless)

TSI: acid slant/ acid deep

Oxidase Test: Negative

IMViC: +-++

Urease Test: Negative

Suceptible to anitbiotics carbapenems, cephalosporin, amikacin

Resistant to gentamicin and tobramycin

Carbapenems, cephalosporin, amikancin
PseudomonasGram positive bacilliMac Conkey: Non-Lactose Fermenting(colourless)

TSI: alkaline slant/ alkaline deep

Oxidase Test: Positive

Catalase Test: Positive

Susceptible to piperacillin, imipenem, tobramycin, ciprolorxacin

Resistant to penicillin

Tobramycin
EnterococcusGram positive cocci

Mac Conkey: Lactose Fermenting(Pink)

Sheep Blood Agar: No Haemolysis

Catalase test: Negative

Resistant to Quinupristin, ampicillin and vancomycinVancomycin

*Mostly Proteus strain positive except P. mirabilis negative.

Urease Test: Positive - Pink, Negative - Orange

Oxidase Test: Positive - Dark Maroon to Black, Negative - Slight pink colouration or no colouration

IMViC: Indole Test, Methyl Red, Voges-Proskauer, Citrate test. Done when oxidase test is negative.

Indole Test: converts tryptophan into indole, ammonia and pyruvic acid. Bacterial culture grown in tryptophan or peptone for 24 to 48 hours before adding kovac reagent. Positive-Red, Negative- Yellow

Methyl Red Test: If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red.

Voges-Proskauer: When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a copper color is present, the result is negative.

Citrate Test: To see if bacteria is able to use only citrate as carbon and energy source. Positive-Blue, Negative- Green.

Reference:

http://www.wikepedia.com/

Douglas
0503224H
TG01

MMIC CASE 4 (2nd posting)

Particulars of patient
Name: Tong Wei Hong
Sex: Male
Age: 68 years

Clinical diagnosis
Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis

Possible Microorganisms

Description

Microscopy



Culture

Biochemical Tests



Streptococcus pneumoniae

- Gram-positive
- Alpha-hemolytic diplococcus bacterium - Fermentative aerotolerant anaerobe - Members of the genus Streptococcus
- Found in the nasopharynx
- Major cause of pneumonia

Gram stain: Gram positive, lancet-shaped diplococcus (stained purple)

1) Blood agar plate (BAP) with added optochin antibiotic disk: alpha-hemolytic (green) colonies and a clear zone of inhibition around the disk

1) Catalase test: Catalase-negative




Haemophilus influenzae

-Non-motile gram-negative coccobacillus
-Found in the upper respiratory system of humans
-Major cause of lower respiratory tract infections, associated with pneumonia

Gram stain: Gram negative coccobacilli, with no specific arrangement (stained pink)

1) Chocolate agar (with factor X (heme) and factor V (NAD)): Flat, greyish-brown colonies colonies with diameters of 1-2mm

1) Catalase test: Catalase-positive

2) Oxidase test: Oxidase-positive



Moraxella catarrhalis

-Gram-negative, non-motile, aerobic, oxidase-positive diplococcus cocci
-Produce beta-lactamase
-Major cause of respiratory tract-associated infection, associated with bronchopneumonia

Gram stain: Gram negative kidney bean shaped diplococcus (stained pink)



1) Enriched media (e.g. Mueller-Hinton, modified Thayer-Martin): Non-pigmented or pinkish-grey opaque colonies

2) Chocolate blood agar: Opaque and non-hemolytic colonies being pushed along the surface of the agar like a hockey puck



1) Oxidase test: Oxidase-positive



2) Catalase test: Catalase-positive



Mycoplasma pneumoniae


-Small bacterium lacking a peptidoglycan cell wall
-Member of the class Mollicutes
-Uniform granular appearance



Nil



Nil



Nil



Chlamydia pneumoniae


-Small bacterium
-Produces round, dense, glycogen-negative inclusion
-Elementary bodies with pear-shaped appearance



Nil



Nil



Nil



Bordetella pertussis

-One of Bordetella species
-Minute gram-negative, aerobic coccobacillus

Gram stain: Gram negative coccobacilli, with no specific arrangement (stained pink)

1) Enriched media- Bordet-Gengou medium (potato-blood-glycerol agar) or BCYE plate that contains penicillin G, using immunofluorescence staining: Mercury-drop colonies
Small, faintly staining gram-negative rods


1) Urease test - Urease-negative

2) Nitrase test - Nitrase-negative

3) Citrate test - Citrate-negative

4) Oxidase test - Oxidase-positive




Possible Microorganisms



Serology



Others





Antibiotic Susceptibility Test



Treatment



Streptococcus pneumoniae



Nil

1) Bile solubility: Colonies soluble in bile

2) Capsule swelling test (Quellung reaction): Capsular swelling in the presence of antisera; Quellung reaction-positive

-Resistant to cephalosporins, macrolides (such as erythromycin), tetracycline, clindamycin and the quinolone

-Susceptible to Vancomycin

Vancomycin in combination with β-lactam antibiotics




Haemophilus influenzae



Nil



Nil

Susceptible to ampicillin, cephalosporins, cefotaxime

Ampicillin (or amoxicillin) if β-lactamase-negative; cefotaxime (IV) or ceftriaxone



Moraxella catarrhalis



Nil

1) Deoxyribonuclease (DNase) test: Presence of DNase

2) Nitrate reduction test: Nitrate and nitrite levels are reduced.

Susceptible to trimethoprim-sulfamethoxazole, fluoroquinolones, cephalosporins, amoxicillin-clavulanic acid

Trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, fluoroquinolones or cephalosporins



Mycoplasma pneumoniae

1) Complement-fixation test: Detects both IgG and IgM specific to M. pneumoniae. A four-fold rise in complement fixation titer during the acute and convalescent phases is indicative of a M. pneumoniae infection.

2) Hemagglutination test: Positive blood test for cold-hemagglutinins
A titer of 1:64 or more indicates infection of M. pneumoniae

3) Enzyme immunoassay: Presence of M. pneumoniae specific IgM

Nil

Susceptible to Macrolide antibiotics, doxycyclin,
second generation quinolones, tetracyclines, erthromycin

Macrolide antibiotics, doxycyclin,
second generation quinolones, tetracyclines, erthromycin



Chlamydia pneumoniae

1) Direct Fluorescent Antibody Test: Detection of IgM or IgG antibodies – a fourfold rise in either the IgM or IgG titers

Presence of proteins (antigens) from Chlamydia pneumoniae



Nil

Susceptible to macrolides, tetracycline, fluoroquinolones, erythromycin,
clarithromycin

Macrolides, tetracycline, fluoroquinolones, erythromycin,
clarithromycin



Bordetella pertussis

1) Direct Fluorescent Antibody (FA) Test: Presence of antibodies
Presence of proteins (antigens) from Bordetella pertussis



Nil

Susceptible to erythromycin

Erythromycin



Pictures of Possible Microorganisms:

Streptococcus pneumoniae on blood agar showing alpha hemolysis (green zone surrounding colonies). Note: the zone of inhibition around a optochin disk.


Streptococcus pneumoniae gram stain of a film of sputum

Streptococcus pneumoniae - Quellung (capsular swelling) reaction

Gram stain of Haemophilus influenzae

Haemophilus influenzae on chocolate agar

Moraxella catarrhalis on chocolate agar


Moraxella catarrhalis on BAP

Gram stain of Moraxella catarrhalis

Electron micrograph of Mycoplasma pneumoniae

Chlamydial inclusions containing pear-shaped C. pneumoniae elementary bodies. EB, elementary body; FG, fatty globules; N, nucleus

Photomicrograph of Bordetella pertussis bacteria using gram staining technique


References

1. http://www.google.com/ > image search > "microorganism name"
2. http://en.wikipedia.org/wiki/Main_Page > "microorganism name"
3. http://www.textbookofbacteriology.net/ > S.pneumoniae
4. Geo.F.Brooks, Janet S.Butel, Stephen A. Morse. (2004). Jawetz, Melnick, & Adelberg's Medical Microbiology Twenty-third Edition. Singapore: The McGraw-Hill Companines, Inc



Dorothy Png
TG01
0503239F

Sunday, December 9, 2007

MMIC PBL Case 2 (2nd Post)

Name: Kwan Siew Yan (outpatient)
Sex: Female
Age: 29
Complaints: Diarrhea
Diagnosis: Enterocolitis
Antibiotic treatment (if any): Nil
Specimen: Stool

Possible Organisms
1) Salmonella enteritidis
2) Shigella species
3) Campylobacter jejuni
4) Staphylococcus aureus
5) Clostridium difficile
6) Bacillus cereus
7) Enteroinvasive E coli (EIEC)

Investigation required:
1) Culture
2) Microscopy
3) Biochemical tests
4) Serology; ELISA and cytotoxicity tests
5) Antibiotic susceptibility tests


Possible Microorganisms

Culture


Microscopy


Biochemical Tests


Salmonella enteritidis


-MacConkey’s agar: non-lactose fermenting (colourless) colonies
-Salmonella-shigella agar: colonies with black centre due to H2S production


-gram-negative rods
- motile


-TSI agar test: alkaline slant and acid butt with gas and H2S
-IMViC tests: gives test result of - + - + respectively.


Shigella species


-MacConkey’s agar: same as Salmonella enteritidi-Salmonella-shigella agar:colonies without black centre as no H2S is produced


-gram-negative rods
- non-motile


-TSI agar test: alkaline slant and acid butt with no gas, no H2S


Campylobacter jejuni


-campylobacter selective media (e.g. Skirrow medium or Campy BAP): grow in scanty amounts at 42 degrees celsius


-curved, gram-negative rods that appear either comma or S-shaped
-motile with a single polar flagellum


-positive oxidase test


Staphylococcus aureus


-Blood agar: yellow or gold colonies


-gram-positive cocci in clusters


-catalase-positive
-coagulase-positive


Clostridium difficile


- Blood agar


- gram-positive
-spore-forming rods


Not done


Bacillus cereus


- Blood agar


- gram-negative
-spore-forming rod


Not done


Enteroinvasive E coli (EIEC)


-MacConkey’s agar: lactose-fermenting colonies


-gram-negative rods


-TSI agar test: alkaline slant and acid butt with gas, no H2S
- IMViC tests: gives test result of + + - - respectively





Possible Microorganisms


Serology; ELISA and cytotoxicity tests


Antibiotic susceptibility tests



Treatment


Salmonella enteritidis


Not done


-resistant to ampicillin and chloramphenicol


-enterocolitis is self limiting in 2-3 days, thus antibiotic treatment is not required.


Shigella species


Not done


-susceptible to ciprofloxacin, ampicillin, doxycycline and trimethoprim-sulfamethoxazole


-fluid and electrolyte replacement without antibiotics or
- one of the following antibiotics: ciprofloxacin, ampicillin, doxycycline and trimethoprim-sulfamethoxazole


Campylobacter jejuni


Not done


-susceptible to erythromycin


- usually symptomatic treatment only; erythromycin for severe disease


Staphylococcus aureus


Not done


- susceptible to penicillin G, nafcillin and vancomycin


-penicillin G for sensitive isolates
-nafcillin is used to treat beta-lactamase resistant strains -vancomycin is used to treat isolates resistant to nafcillin


Clostridium difficile


-Exotoxin in stool detected by cytopathic effect on cultured cells. Identified by neutralization of cytopathic effect with antibody
-Assessment of the A and B toxins in stool can be detected by using ELISA


-susceptible to metronidazole


-metronidazole. Vancomycin, although effective, should not be used as it may select for vancomycin-resistant enterococci


Bacillus cereus


Not done


-susceptible to vancomycin, chloramphenicol, gentamicin, tetracyclines or sulfonamides


-only symptomatic treatment is given antibiotics such as vancomycin, chloramphenicol, gentamicin, tetracyclines or sulfonamides


Enteroinvasive E coli (EIEC)


Not done


- susceptible to trimethoprim-sulfamethoxazole, aminoglycoside, ampicillin, cephalosporin and sulfonamides


- treatment can be difficult because of multiple drug resistance as mediated by ``R’’ plasmid




MacConkey’s agar is a selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies. [2]

Salmonella-shigella agar are designated as moderately selective media based upon the degree of inhibition of gram-positive microorganisms that they inhibit due to their content of bile salts, brilliant green and citrates. [3] This media is used to find out whether the suspected microorganism is either Salmonella enteritidis or Shigella sp.

Skirrow’s agar is peptone and soy protein base agar supplemented with lysed horse blood and vancomycin (inhibits Gram-positives), polymyxin B (antifungal), and trimethoprim (broad spectrum). Campy BAP is brucella agar supplemented with sheep blood and vancomycin, trimethoprim and cephalothin (inhibits streptococci) [4]

Blood agar is an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. β-hemolytic activity will show complete lysis of red blood cells surrounding colony, while α-hemolysis will only partially lyse hemoglobin and will appear green. γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity. [2].

After the microorganisms had been isolated, microscopy is used to observe the morphological characteristics so that to identify the organism. Gram stain is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. Gram-positive bacteria have cell wall made of peptidoglycan which stains purple and Gram-negative bacteria which stain pink. [5]. Spore stain will be required to identify spores. Wet mount microscopy is used initially before isolation of organisms to identify their motility.

Antibiotic Susceptibility Test, also known as Kirby-Bauer test, is performed by swabbing the bacterium on the agar and the antibiotic discs are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. If the organism is killed or inhibited by the concentration of the antibiotic, there will be no growth in the immediate area around the disc. This is called the zone of inhibition. The zone sizes are looked up on a standardized chart to give a result of sensititive, resistant, or intermediate. [6] This can help to provide the necessary treatments for the patient.

Pictures
Salmonella enteritidis on Salmonella-shigella agar
[7]


[8]


Staphylococcus aureus (gram stain)
[9]

Campylobacter jejuni wet mount microscopy[10]


Clostridium difficile (gram stain)[11]


Bacillus cereus on blood agar[12]


Enteroinvasive E coli (EIEC)on MacConkey agar[13]













References
1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
2) http://en.wikipedia.org/ >Agar_plate
3) http://www.bd.com/ >SS_Agar
4) http://www.life.umd.edu/ >Media
5) http://en.wikipedia.org/ >Gram_stain
6) http://www.rlc.dcccd.edu/ >antibiotics
7) http://www.rci.rutgers.edu/ >salmonellashigella
8) http://textbookofbacteriology.net/ >Shigella
9) http://en.wikipedia.org/ >Staphylococcus_aureus
10) http://www.umm.edu/ >fecal culture
11) http://en.wikipedia.org/ >Bacillus_cereus
12) http://www.spiceisle.com/ > Enteric demonstration

Soong Ci Liang
TG01
0503333G

MMIC laboratory diagnosis

Patient particulars:

Name: Wong Fei Hong

Sex: Male

Age: 37 years

Complaints: fever sewlling around operation wound

Diagnosis: wound infection

List of possible organismTest requiredExpected resultstreatment
Corynebacterium diphtheriae

1) Gram stain

2) Methylene blue

3) Culture (Loffler medium followed by tellurite plate)

4) PCR assay

1) gram-positive, highly pleomorphic organisms with no particular arrangement

2) Demostrate the presence of metachromatic granules

3) If C. diphtheriae is presence, tellurium in the medium will be reduced to elemental tellurium by the organism, showing a grap-black color of tellurium in the colony

4) Identify the toxin gene presence in the organism isolated form the patient

Antitoxin

penicillinG

Erythromycin

Pseudomonas aeruginosa

1) Gram stain

2) Culture (EMB or MacConkey’s agar)


3) TSI medium

4) Biochemical test (catalase, oxidase, nitrase, lipase)

1) Gram negative rod with no particular arrangement

2) Culture of patient specimen on MacConkey’s agar produce colourless colonies as the organism does not ferment lactose

3) Alkaline slant and an alkaline butt as the organism does not ferment lactose

4) Catalase, Oxidase, Nitrase and Lipase positive

Antipseudomonal

penicillin


Aminoglycoside

staphylococcuss aureus

1) Gram stain

2) culture (Mannitol salt agar)

3) coagulase test

4)biochemical test (catalase test)

1) Gram positive cocci in grapelike clusters

2) Golden yellow colonies

3) coagulase positive

4)catalase positive

Mupirocin

Beta-lactamose resistant penicillins such as nafcillin, cloxacillin, cephalosporins or vancomycin


Proteus mirabilis

1) Gram stain

2) culture (EMB or MacConkey’s agar)3) TSI agar

4) biochemical test (indole test

1) Gram negative rod

2) Non-lactose fermenting colonies on EMB or MacConkey’s agar

3) alkaline slant and acid butt.

4) indole negative

Ampicillin

Tetracycline

group A streptococcus bacterium

1) Gram stain

2) Culture (blood culture)

3) Biochemical test (indole test)

1) Gram positive cocci in chain

2) Beta hemolytic colonies

3) Indole test negative

Penicillin G

Bacteroides fragilis

1) Gram stain

2) Anaerobic Culture blood agar plates containing kanamycin and vaomyycin

3) TSI agar

1) Gram negative rod

2) Colonies should be able to grow on the BAP containing the anitbiotic

3) Red stunt yellow butt

Penicillin G

Cephalosporins

Aminoglycosises

Metronidazole

Pasteurella Multocida

1) Gram stain

2) Culture (blood agr or chocolate agar)

3) oxidase test

4) catalase test

1) Encapsulated gram negative rod

2) positive

3) positive

4)positive

Penicillin G

Aeromonas Hydrophila

1) Gram stain

2) Culture (Sheep blood agar with ampicillin)

3) catalse test

4) oxidase test

1) Gram negative rod

2) Aeromonas Hydrophila is able to grow on the agar plate while the ampicillin will inhibit the grow of other competing organism

3) positive

4) positive

chloramphenicol
Clostridium perfringens

1) Gram stain

2) Culture ( blood agar)

1) Large gram postitive rods

2) Exhibit a double zone of hemolysis

Penicillin G

Types of wound infection and their causative agents:

-Skin infection at the site of preexisting skin leison: Corynebacterium diphtheriae

-Burn wound infection: Pseudomonas aeruginosa

-Wound infection in diabetic patients: Staphylococcuss aureus

-Surgical wound infection: Staphylococcuss aureus

-Abcess infection of brain, lung or abdominal: Bacteroides fragilis

-Wound infection caused by cats or dog bite: Pasteurella multocida

-Traumatic open wound infection: Aeromonas hydrophila

-Gas gangrene wound infection: Clostridium perfringens

Reference:

1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

Wing Fat

Tg01

Friday, December 7, 2007

Compilation of All Potential Microorganisms To Each Case










































































Diagnosis Potential PathogensDescriptions
Case 1 Urinary Tract Infection (UTI)
1) Escherichia Coli
2) Klebsiella spp.
3) Streptococcus
spp. (GPC)
4) Staphylococcus spp. (GPC)
Eg. S saprophyticus, Staphylococcus aureus
5) Pseudomonas spp.
6) Proteus spp.
7) Candida spp.
Eg. Candida albicans
8) Enterococcus
9) Morganella
10) Serratia

1) Gram-negative bacilli

2) Gram-negative bacilli

3) Gram-positive cocci

4) Gram-positive cocci

5) Gram-positve bacilli

6) Gram-negative bacilli

7) Gram-negative bacilli

8) Fungus

9) Gram-positive cocci

10) Gram-negative bacilli

11) Gram-negative bacilli
Case 2 Enterocolitis
1) Salmonella enteritidis

2) Shigella species

3) Campylobacter jejuni

4) Staphylococcus aureus

5) Clostridium difficile

6) Bacillus cereus

7) Enteroinvasive Escherichia coli (EIEC)


1) Gram-negative rods

2) Gram-negative rods

3) Gram-negative rods

4) Gram-positive cocci in clusters

5) Gram-positive rods

6) Gram-negative rods

7) Gram-negative rods
Case 3 Urinary Tract Infection (UTI)
1) Escherichia Coli

2) Proteus spp.

3) Klebsiella spp.

4) Serratia

5) Proteus

6) Pseudomonas spp.

7) Streptococcus spp.

8) Enterococcus

9) Candida spp. (Fungus)

1) Gram negative bacilli
2) Gram negative bacilli
3) Gram negative bacilli
4) Gram negative bacilli
5) Gram negative bacilli
6) Gram positive bacilli
7) Gram positive cocci
8) Gram positive cocci
9) -
Case 4 Bronchitis

1) Streptococcus pneumoniae

2) Haemophilus influenzae

3) Moraxella catarrhalis


1) Gram-positive cocci

2) Gram-negative coccobacillus

3) Gram-negative diplococci
Case 5 Wound Infection
1) Corynebacterium diphtheriae

2) Pseudomonas aeruginosa

3) Staphylococcuss aureus

4) Proteus mirabilis

1) Gram positive

2) Gram negative rod

3) Gram positive coccus

4) Gram negative rod
Case 6 Urinary Tract Infection (UTI)
1) Escherichia coli

2) Proteus

3) Chlamydia

4) Gonorrhea

5) Herpes simplex virus type 2 (HSV-2)

6) Candida

7) Parasites

Monday, December 3, 2007

MMic PBL Case 3

MMic PBL



Name: Maisy Hong

Sex: Female

Age: 67

Complaints: Fever, chills, bladder distension; on indwelling catheter
(Bladder distention on indwelling catheter: this refers to implanting a flexible or rigid hollow tube to distend body passages, or to drain fluids from body cavities. [1] )


Diagnosis: Urinary Tract Infection
(A urinary tract infection, or UTI, is an infection that can happen anywhere along the urinary tract. The urinary tract includes the bladder, kidneys, ureters and urethra.[2])

Antibiotic treatment (if any): Nil

Specimen: Urine


GramShapePossible Microorganism
NegativebacilliEscherichia Coli
NegativebacilliProteus spp.
NegativebacilliKlebsiella spp.
NegativebacilliSerratia
NegativebacilliProteus
PositivebacilliPseudomonas spp.
PositivecocciStreptococcus spp.
PositivecocciEnterococcus
--Candida spp. (Fungus)


Identification of Microorganism



  1. Gram staining and microscopy.
  2. Catalase Test.
  3. Oxidase Test.
  4. Biochemical Test.
  5. Aerobic culture on sheep blood agar, Cysteine Lactose Electrolyte Deficient Medium, and Mac Conkey agar.
  6. Antibiotics sensitivity testing.

Reference

[1] http://dictionary.reference.com/
[2]MedlinePlus. (2007) Urinary Tract Infection. Retrieved December 02, 2007, from http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm>urinary tract infection

Douglas
TG01

Sunday, December 2, 2007

MMIC

Name: Wong Fei Hong
Sex: Male
Age: 37
Complaints: Fever, swelling around operation wound
Specimen: Wound swab
Diagnosis: Wound infection

A wound infection happens when pathogens enter an open wound of the patient. Factors such as disease, surgery and weak immune system increase the patient’s risk of getting wound infection. Signs and symptom of a wound infection includes:
-Redness or swelling around the wound
-Secretion of Pus or watery discharge around the wound area
-foul odour from the wound
-mild fever

Possible pathogens that cause wound infection:
-Corynebacterium diphtheriae
-Pseudomonas aeruginosa
-staphylococcuss aureus
-Proteus mirabilis


Corynebacterium diphtheriae: C. diphtheriae is a facultative anaerobic, gram positive organism that can infect skin lesions in people with poor skin hygiene, causing cutaneous diphtheria. Symptom of cutaneoud diphtheria includes mild fever and swollen wound.

Pseudomonas aeruginosa: Pseudomonas aeruginosa is an aerobic, gram negative rod shape bacterium that can cause burn infection and wound infection. The microorganism is able to produce endotoxin that causes fever.

Staphylococcuss aureus: Staphylococcuss aureus is a facultative, gram positive coccus organism that causes localised infection in forms of abscess and skin infection, which is one of the possible causes to surgical wound infections.

Proteus mirabilis: Proteus mirabilis is A facultative, gram negative rod proteus species that cause uriniry tract infection, but it can also cause wound infection, mostly in hospitalized patients.

Laboratory diagnosis:
-Gram stain
-Culture (aerobic/anaerobic)
-biochemical reactions
-oxidase tests
-catalase tests
-antibiotic susceptibility

Wing Fat
Tg01

MMIC PBL

Particulars of patient
Name: Tong Wei Hong
Sex: Male
Age: 68 years

Clinical diagnosis
Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis

Bronchitis is inflammation of the large airways that branch off the trachea (bronchi), usually caused by infection but sometimes caused by irritation from a gas or particle.


In bronchitis, areas of the bronchial wall become inflamed and swollen, and mucus increases. As a result, the air passageway is narrowed. Bacteria and viruses are usually present.

There are two main types of bronchitis:

-Acute bronchitis comes on rapidly, usually after a virus has invaded the upper respiratory tract. Sometimes there is a bacterial infection as well. Viruses most likely to trigger acute bronchitis are those responsible for influenza (the flu) or the common cold.

-Chronic bronchitis is defined as excessive mucus secretion in the bronchi and a chronic or recurrent mucus-producing cough that lasts three or more months and recurs year after year. People who have chronic bronchitis are more susceptible to bacterial infections of the airway and lungs, like pneumonia.

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.

Viral bronchitis may be caused by a number of common viruses, including the influenza virus (influenza A and B). A person often has a combination of bacterial and viral bronchitis.

Possible causative agents

The below 3 microorganisms often cause bacterial bronchitis in middle-aged and older people.

Possible Microorganisms

Description

Streptococcus pneumoniae

-Gram-positive
-Alpha-hemolytic diplococcus bacterium
-Members of the genus Streptococcus
-Found in the nasopharynx
-Major cause of pneumonia

Haemophilus influenzae

-Non-motile gram-negative coccobacillus
-Found in the upper respiratory system of humans
-Major cause of lower respiratory tract infections, associated with pneumonia

Moraxella catarrhalis

-Gram-negative, aerobic, oxidase-positive diplococcus cocci
-Major cause of respiratory tract-associated infection, associated with bronchopneumonia


Investigation required

Specimen: Sputum
*Clear or white sputum suggests a viral infection; yellow or green sputum suggests a bacterial infection.

  • Microscopy
    - Gram stain (Gram positive, -negative)

  • Culture
    - Aerobic on blood agar plates
  • Antibiotic Suspectibility
  • Serology (detection of viruses)
    - Haemagglutinin Inhibition (HI)
    - Enzyme-Linked ImmunoSorbent Assay (ELISA)
  • Others
    - Oxidase test (Oxidase-positive, -negative)
    - Catalase test (Catalase-positive, -negative)

References

1) http://www.merck.com/mmhe/sec04.html > Bronchitis
2) http://en.wikipedia.org/wiki/Main_Page > Moraxella_catarrhalis
3) http://en.wikipedia.org/wiki/Main_Page > Haemophilus_influenzae
4) http://en.wikipedia.org/wiki/Main_Page > Streptococcus_pneumoniae

Dorothy
TG01

MMIC PBL

Name: Kwan Siew Yan (outpatient)
Sex: Female
Age: 29
Complaints: Diarrhea
Diagnosis: Enterocolitis
Antibiotic treatment (if any): Nil
Specimen: Stool

Enterocolitis is the inflammation of the large and small intestines. [1] It is characterized by an invasion of the epithelial and subepithelial tissue of the small and large intestines. Strains that do not invade do not cause this disease. The organisms penetrate both through and between the mucosal cells into the lamina propria, resulting in inflammation and diarrhea. A polmorphonuclear leukocyte will limit the infection to other parts of the body. [2] Symptoms will include:
- diarrhea
- nausea
- vomiting
- fever
- abdominal bleeding
- rectal bleeding
- sluggishness [1]

Possible Organisms Present [2]:
1) Salmonella enteritidis
Characteristics
- facultative anaerobes
- gram-negative rods
- do not ferment lactose
- produce H2S
- motile

Habitat and Transmission
- found in enteric tract of humans and animals
- transmitted mainly by fecal-oral route and foods prepared by chronic carriers

Pathogenesis
- invade the mucosa of the small and large intestines
- infectious dose is at least 10*5 organisms, much greater than infectious dose of Shigella

2) Shigella species
Characteristics
- facultative gram-negative rods
- do not produce H2S
- non-lactose fermenting
- non-motile compared to Salmonella

Habitat and Transmission
- human colon only; human disease
- no animal reservoir for Shigella
- transmitted by the fecal-oral route
- principal factors in transmission include fingers, flies, food and feces

Pathogenesis
- invade the mucosa of the ileum and colon but do not penetrate further; hence sepsis is rare
- most effective pathogens among enteric bacteria as infectious dose is much lower (1-10 organisms) than that of Salmonella

3) Campylobacter jejuni
Characteristics
- curved, gram-negative rods that appear either comma or S-shaped
- motile with a single polar flagellum
- microaerophilic (5% O2 with 10% CO2)
- grow well at 42℃

Habitat and Transmission
- human and animal feces
- transmitted by fecal-oral route
- Food and water contamination with animal feces is the major source of human infection. (e.g. poultry, meat and unpasteurized milk)

Pathogenesis
- invade mucosa of colon but do not penetrate; hence sepsis is rare

4) Staphylococcus aureus
Characteristics
- gram-positive cocci in clusters
- coagulase-positive and catalase-positive

Transmission
- via the hands

Pathogenesis
- produce enterotoxin in foods

5) Clostridium difficile
Characteristics
- anaerobic
- gram-positive
- spore-forming rods

Habitat and Transmission
- human colon and transmitted by fecal-oral route

Pathogenesis
- antibiotics suppress normal flora of colon, allowing them to overgrow and produce large amounts of exotoxins

6) Bacillus cereus
Characteristics
- aerobic
- gram-negative
- spore-forming rod

Habitat and Transmission
- found in grains such as rice (e.g. re-heated rice)

Pathogenesis
- Two enterotoxins are produced

7) Enteroinvasive Escherichia coli (EIEC)
Characteristics
- Facultative gram-negative rods
- Ferment lactose

Transmission
- Fecal-oral route
- Food-borne route

Pathogenesis
- closely resemble Shigella in their pathogenic mechanisms
- penetrate and multiply within epithelial cells of the colon causing widespread cell destruction
- EIEC are invasive organisms which are same as Shigella but they do not produce LT or ST toxin and, unlike Shigella, they do not produce the shiga toxin

References

1) http://www.wrongdiagnosis.com/ >enterocolitis

2) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company.

Soong Ci Liang
0503333G
TG01

MMIC PBL

Particulars of Patient

Name: Ong Fei Fei
Sex: Female
Age: 37

Clinical Diagnosis

Complaints: Fever, pain during urination, virginal discharge
Diagnosis: UTI
Antibiotic Treatment: Nil
Particulars of Specimens
Specimen: Vaginal Discharge

Description of Urinary Tract Infection (UTI)

In healthy people, urine in the bladder is sterile—no bacteria or other infectious organisms are present. The channel that carries urine from the bladder out of the body (urethra) contains no bacteria or too few to cause an infection. However, any part of the urinary tract can become infected; an infection anywhere along the urinary tract is called a urinary tract infection.

UTIs are usually classified as upper or lower according to where they occur along the urinary tract. Lower UTIs are infections of the urethra (urethritis) or bladder (cystitis); upper UTIs are infections of the kidneys (pyelonephritis) or ureters (ureteritis).

Causes

UTIs are almost always caused by bacteria, although some viruses, fungi, and parasites can infect the urinary tract as well. Common organisms that cause UTIs include: Escherichia coli and Staphylococcus saprophyticus. Less common organisms include Proteus mirabilis, Klebsiella pneumoniae, Enterobacter spp, Pseudomonas and Enterococcus spp.

Bacteria: Bacterial infections of the lower urinary tract—the bladder and urethra—are very common. Escherichia coli is the most common bacteria. When the person has a kidney stone, Proteus bacteria may be able to grow. These conditions can lead to abnormal vaginal discharge which can be caused by Chlamydia and Gonorrhea.

Viruses: The herpes simplex virus type 2 (HSV-2) may infect the urethra to cause a UTI, making urination painful and emptying of the bladder difficult.

Fungi: Certain fungi or yeasts can infect the urinary tract to cause a UTI. The most common type is Candida, which causes candidiasis.

Parasites: A number of parasites, including certain types of worms, can infect the urinary tract.

Trichomoniasis, caused by a type of microscopic parasite, is a sexually transmitted disease that can produce a copious greenish yellow, frothy discharge from the vagina in women.


Investigation required

Microscopy, culture of specimen and antibiotic susceptibility should be requested.

Doreen Ng (tg 01)

MMIC PBL Case 1

Subject: MMIC
Case: Case 1 - Khong Fay Seah
Complaints: Fever, chills, dysuria,
Diagnosis: Urinary Tract Infection

Urinary Tract Infection: A urinary tract infection, or UTI, is an infection that can happen anywhere along the urinary tract. The urinary tract includes the bladder, kidneys, ureters and u rethra. [1]

Dysuria: Painful or difficult urination. This includes burning on urination. Dysuria is most commonly due to bacterial infection of the urinary tract causing inflammation of the bladder (cystitis) or kidney (pyelonephritis).
In women, dysuria may also reflect inflammation of the vagina (vaginitis) or vulva (vulvitis). And in men, dysuria may be due to inflammation of the prostate (prostatitis) or the urethra (urethritis) from gonorrhoea or chlamydia. [2]

Possible Pathogens

Possible MicroorganismsDescriptions
Escherichia ColiGram-negative bacilli
Klebsiella spp.Gram-negative bacilli
Streptococcus spp. (GPC)Gram-positive cocci
Staphylococcus spp. (GPC)
Eg. S saprophyticus, Staphylococcus aureus
Gram-positive cocci
Pseudomonas spp.Gram-positve bacilli
Proteus spp.Gram-negative bacilli
Candida spp.
Eg. Candida albicans
Fungus
Enterococcus faecalisGram-positive cocci
MorganellaGram-negative bacilli
SerratiaGram-negative bacilli



Investigation Required
Microscopy:
1) Gram stain - Differentiate Gram positive and Gram negative bacteria
2) Wet mount and KOH smear - Demonstration of pseudohyphae for identification of Candida spp.

Culture:
Aerobic culture on Blood plate agar(BAP), Cysteine Lactose Electrolyte Deficient medium (CLED), MacConkey (MAC)agar plate.

Other Tests:
1) Catalase Tests
2) Oxidase Tests
3) Biochemical Tests

Antimicrobial Susceptibility Test:
To determine the treatment

References:
1. MedlinePlus. (2007) Urinary Tract Infection. Retrieved December 02, 2007, from http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm
http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm > >urinary tract infection
2. MedicineNet, Inc. (2007) Definition of Dysuria. Retrieved December 02, 2007, from http://www.medterms.com/script/main/art.asp?articlekey=3163
http://www.medterms.com/script/main/hp.asp > dysuria

Boon Ching
Tg01

Sunday, November 11, 2007

Treponemal Test – TPPA

Introduction
The Treponema pallidum particle agglutination (TP-PA) test is a treponemal test (confirmation test) for the serologic detection of antibodies to the various species and subspecies of pathogenic Treponema, the causative agents of syphilis, yaws, pinta, bejel, and endemic syphilis. The test is a passive agglutination procedure based on the agglutination of gel particles sensitized with T. pallidum antigens by antibodies found in the patient's serum (1-3). The test is intended as a confirmatory test to replace the microhemagglutination assay for antibodies to T. pallidum (MHA-TP).

Principle
Serum containing antibodies to pathogenic treponemes react with gel particles sensitized with sonicated T. pallidum, Nichols strain (the antigen), to form a smooth mat of agglutinated gel particles in the microtiter tray well.

If antibodies are not present, the particles settle to the bottom of the tray well, forming a characteristic compact button of unagglutinated particles.

The unsensitized gel particle control well for each serum should also show this compact button, or the absence of agglutination.

The TPPA test is used to confirm the reactive results of a nontreponemal screening test for syphilis, such as the VDRL slide test, or as a diagnostic test in patients with a nonreactive nontreponemal test but with signs or symptoms suggestive of late syphilis

Interpretation of results
1) Read the settling patterns of the gelatin particles using an angled mirror (tray viewer) to visualize the patterns from below - Readings are scored on a scale of – to 2+ agglutination
2) Retest serum with samples with a reading of ±

Boon Ching
TG01

Sunday, November 4, 2007

special stain

Martius scarlet blue (MSB)

Function: stain fibrin

Principle: the main feature of MSB is the use of a small molecule yellow dye, together with phosphotungstic acid to selectively stain red cells. Phosphotungstic acid blocks the staining of muscle, collagen and most connective tissue fibres. Muscle and fibrin are stained with medium size molecule red dye, while staining of collagen is prevented by phosphotungstic acid. Larger blue dye will then stained the collagen and fibrin.

Control: skeletal or cardiac muscle, soft cartilage, fibrinous appendix

Chemical required: 1% acetic acid
2%phosphotungstic acid
martius blue
martius red
martius yellow
weigert iron haematoxylin

Reagent preparation: martius scarlet blue working solution
martius yellow 1 part
martius blue 2 parts
martius red 3 parts

Procedure:
1) dewax, section to water
2) leave slide overnight in zenker’s solution
3) run in water until colourless
4) stain with weigert iron haematoxylin for 10 minutes
5) blue in water
6) stain in martius scarlet blue working solution for 10 mins
7) wash with acetic acid
8) dehydrate, clear, mount

Result: nuclei: blue/black
Erythrocyes: yellow
Fibrin: red
Connective tissue: blue


Wing Fat
TG01

Biochemistry

For the past two weeks, i am attached to biochemistry laboratory. In this lab, there are quite a number of tests they perform. For example, Urinanalysis, Fungal smear, occult blood, liver function tests, lipid panel and others. Here, in this entry, i will like to discuss about neonatal total bilirubin test and HbA1C.

Neonatal total bilirubin

This test is to screen for and monitor liver disorders, such as jaundice, or liver diseases, such as cirrhosis.

A bilirubinometer which is a direct spectrophotometer is used to measure the total bilirubin in neonatal serum to aid in indicating the risk of bilirubin encephalopathy.

Procedures
  1. Centrifuge the capillary tube of neonatal blood for 3 minutes at 3000rpm.
  2. By using a pipette, completely fill the cuvette with serum, holding the narrow portion of the cuvette between your thumb and index finger.
    (To avoid erroneous readings, the outside surface of the filled cuvette must clean. The sample seen in the clear rectangular area of the cuvette must be free of bubbles. If bubbles are observed, tap the cuvette lightly to clear them and refill as necessary.)
  3. Open the black door on the top of the instrument and insert the cuvette into the sample carrier until the widest portion of the cuvette is seated. Close the door.
  4. Press the START button. There will be a 2 second delay to allow the lamp to come to equilibrium, after which the sample carrier will rotate to bring the sample cuvette into the optical path. After a short pause, bilirubin concentration will be displayed.
  5. After 15 seconds, the measurement cycle will be complete and the sample carrier will return the sample to the load position. Remove the cuvette.
  6. The display will show the bilirubin value for approximately 10 seconds, then clear.

Once the result is read from the display, we will record it down on the patient’s requisition form and send it for verify.

Interpretation of results

  • Newborns: When bilirubin levels are high, a condition called jaundice occurs, and further testing is needed to determine the cause. Too much bilirubin may result from the rupture of the red blood cells, or that the liver is incapable of removing bilirubin from the blood. It is not uncommon to see high bilirubin levels (sometimes called neonatal bilirubin) in newborn babies (typically 1–3 days old). Within the first 24 hours of life, up to 50% of full-term newborns, and an even greater percentage of pre-term babies, may have high bilirubin level. At birth, the newborn lacks the intestinal bacteria that help process bilirubin. This is not abnormal and resolves itself within a few days. In other instances, newborns’ red blood cells may have been destroyed because of Rhesus blood typing incompatibility.
  • Adults and children: In adults or older children, bilirubin is measured to diagnose and/or monitor liver diseases (such as cirrhosis, hepatitis, or gallstones).

HBA1c

HbA1c is a test that measures the amount of glycosylated hemoglobin in your blood. It is used to measures your blood sugar control over several months and gives a good estimate of how well diabetes is being managed over a period of weeks to months.

Glycosylated hemoglobin (hemoglobin A1c, Hb1c, HbA1c or HgA1c) is a molecule in red blood cells that attaches to glucose (blood sugar). It is a form of hemoglobin used primarily to identify the average plasma glucose concentration over prolonged periods of time. Its name is sometimes abbreviated to A1C. It is formed in a non-enzymatic pathway by hemoglobin's normal exposure to high plasma levels of glucose. Glycation of hemoglobin has been implicated in nephropathy and retinopathy in diabetes mellitus.

Monitoring the HbA1c in diabetic patients may improve treatment.

Normal Results

HbA1c is normal if it is 5% or less. Normal ranges may vary slightly.

Abnormal Results

Abnormal results mean that your blood glucose levels have not been well-regulated over a period of weeks to months. If your HbA1c is above 7%, it means your diabetes is poorly controlled.

That's all for this entry. hope u enjoy reading. =)

Doreen Ng (tg 01)



Sunday, October 28, 2007

SIP

Subject: Blood Banking
Topic: Bone Marrow Transplantation (BMT)

Bone marrow contains immature cells (stem cells), which develops into RBCs, WBCs & platelets

Purpose: To replace unhealthy cells with healthy ones in order to survive
Patients: People with diseases of the blood, bone marrow, or certain types of cancer, (e,g., Hodgkin's disease, mulitple myeloma, leukaemia, etc.)

Conditioning/preparative regimen: Treatment with high doses of chemotherapy or total body irradiation, given immediately prior to a transplant.
Purpose: To help remove the patient's disease prior to the BMT & to suppress immune reactions. It also has an immunosuppressive effect which prevents rejection of the BMT by the recipient's immune system.
If donor's bone marrow doesn not match, it'll treat the patient's body as foreign material to be attacked & destroyed, which results in graft vs host disease (GVHD). This is when patient's own immune system destroy the new bone marrow (graft rejection).

During the period when patient waits for the transplanted bone marrow to migrate to the cavities of large bones, setting up 'engraft' to the stage where it begins to produce normal blood cells, the patient is susceptible to infection & excessive bleeding. Platelets are transfused to patients who are bleeding.

Until the donor marrow engrafts, RBCs & platelets will be needed to keep the hemoglobin level above 9g/dl and platelet count > 20,000/ul. Engraftment takes place in 2 to 4 weeks but may be delayed if complications occur.

As a result of the total body irradiation & chemotherapy required for preparation for transplantation, patients are immunosuppressed & susceptible to GVHD, if transfused with blood components that contains live lymphocytes, which are capable of dividing. Tranfusion related GVHD can be prevented by irradiating all blood components.
Why use irradiated & filtered (leucodepleted) blood products?
To prevent patients from febrile non-hemolytic transfusion reaction due to leucocytes & leucocytes fragments in whole blood or packed cells.
Blood cell gamma irradiator is used for irradiating blood components such as red cell or platelet concentrates. The gamma rays effectively stop cells dividing by mitotic division, preventing lymphocyte proliferation.

Since alloimmunisation is caused by leucocytes contained in the blood components, leucodepleted red cells has been used to minimise the likelihood of inducing alloimmunisation.
The immunosuppression also results in great susceptibility to infectious diseases. Most patients will receive anitbiotics, antifungal agents & intravenous immunoglobulin to help prevent fight infection.

ABO Incompatible Transplants:
ABO incompatible transplants may be done when there is patient antibody directed against donor cells (major incompatibility; e.g., patient O, donor A) or donor antibody directed against the patient's red cells (minor incompatibility; e.g., patient A, donor O).

Example 1:
Patient 1's blood group: A+
Donor 1's blood group: O+

Blood product (irradiated & filtered): Packed cell: O+
Platelet/CSP: Group A+ (1st choice)/Group O+

Problem: Continued production of antibody by the new donor lymphocytes while there are original patient's cells continuing to circulate. Thus, the group O donor cells may begin to produce anti-A while the orginal patient A red cells remains, leading to hemolysis.
Solution: This can be lessened by red cell exchange transfusion if necessary.

Group A Recipient with Group O Donor:
Anti-AAnti-BAnti-ABA CellsB CellsO CellsInterpretation
+-+-+-Group A
+/----+-Incomplete Group A
---+/-+-Incomplete Group O
---++-Complete Group O

ABO blood grouping of transfused patient:
For incomplete group A, there will be a mixed field reaction when tested with anti-A. Some of the red cells are agglutinated while others are not. The anti-sera will appear to be cloudy due to some free red cells. This occurs when the patient's blood group (A+) is slowly switching to the new donor's blood type (O+).

Example 2:
Patient 2's blood group: O+
Donor 2's blood group: A+

Blood product (irradiated & filtered): Packed cell: O+
Platelet/CSP: Group A+ (1st choice)/Group AB+

Problem: When ABO-incompatible marrow is to be transfused into a patient with circulating antibodies against the donor's ABO type, hemolysis of the red cells in the marrow can be expected.
Prevention: By processing the marrow to remove the red cells.
Following transplant & after engraftment, the patient will become A+ but cannot begin to receive group A red cells until the circulating anti-A has disappeared. Anti-A persists longer than anti-B. Thus, even after transfusion of type A marrow, the patient continues to receive type O red cells containing additonal anti-A that can slow down the appearance of type A red cells.
If delayed red cell engraftment is occurring, the titer of anti-A should be determined & a DAT done to determine if high levels of circulating anti-A are hemolysing newly forming A red cells.
In making the decision when to convert to the new donor ABO type for red cell transfusion, we use the crossmatch to determine the absence of circulating ABO antibody incompatible with the patient's original type. The patient's serum should be tested in the antiglobulin phase to ensure the absence of free ABO-incompatible antibody.
When the ABO antibody to donor type is undetectable & DAT is negative, this is a satisfactory indicator of the safety to begin transfusion the new donor ABO type red cells.

Group O Recipient with Group A Donor:
Anti-AAnti-BAnti-ABA CellsB CellsO CellsInterpretation
---++-Group O
+/--+/--+-Incomplete Group A
+-+-+-Complete Group A


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Thursday, October 18, 2007

Staining and staining problems

Staining and staining problems

There is 2 main types of stains that is used most commonly. They are the Romanowsky and Papanicolaou(and it's derivatives). Romanowsky-type stain are more rewarding and practical, and readily available in practice situations. Example of such stains are Wright's stain, Giemsa stain, Diff-Quik. Romanowsky-type stain are inexpensive, readily available to practicing veterinarian, easy to prepare, maintain and use. They stain organism and cytoplasm of cells very well.

Smears are first air dried, preserving(fix) the cells, and cause them to adhere to the slide so they do not fall off during the staining procedure. Diff-Quik does not undergo the metachromatic reaction. As a result, granules of some mast cells do not stain, When mast cell granules do not stain, the mast cells may be misclassified as macrophages. This can lead to confusion in examination of some mast-cell tumors. Variation between different Romanowsky-type stains should not cause a problem once the evalutator has become familiar with the stain used routinely.

The procedure recommended for each stain should be followed generally. However, adaptations is to be applied according to the thickness of smear. Thick smear, higher protein concentration, longer stain time. Vice-versa for thin smear.

Poor stain quality often confuse both the novice and the experienced cytologist. Most staining problem can be avoided if the following precaution are taken:

  • Use new slides, fresh and well filtered(if needed) stain, and fresh buffer(if needed)

  • Stain cytologic preparations immediately after drying

  • Take care not to touch the surface of slide or smear at any time.

The table below shows some common problems and solution.

ProblemSolution
Excessive Blue Staining
Prolonged stain contactDecrese staining time
Inadequate washWash longer
Specimen too thickMake thinner smears
Stain, diluent, buffer or wash water too alkalineCheck with pH paper and correct pH
Exposure to formalin vaporsStore and ship cytologic preps separate from formalin containers
Wet fixation in ethanol or formalinAir dry smears before fixation
Delayed fixationFix smears sooner
Surface of the slide was alkalineUse new slides
Excessive Pink Staining
Insufficient staining timeIncrease staining time
Prolonged washingDecrease duration of wash
Stain or diluent too acidicCheck with pH paper and correct pH; fresh methanol may be needed
Excessive time in red stain solutionDecrease stain time in red solution
Inadequate time in blue stain solutionIncrease time in blue stain solution
Mounting coverslip before preparation is dryAllow preparation to dry completely before mounting coverslip
Weak Staining
Insufficient contact with one or more of the stain solutionsIncrese staining time
Fatigued(old) stainsChange stains
Another slide coverd specimen during stainingKeep slides separate
Uneven Staining
Variation of pH in different areas of slide surfaceUse new slides and avoid touching their surface before and after preparation
Water allowed to stand on some areas of the slide after staining and washingTilt slides close to vertical to drain water from the surface or dry with a fan
Inadequate mixing of stain and bufferMix stain and buffer thoroughly
Precipitate on Preparation
Inadequate stain filtrationFilter or change the stains
Inadequate washing of slide after stainingRinse slides well after staining
Dirty slides usedUse clean new slides
Stain solution dries during stainingUse sufficient stain and do not leave it on slide too long
Miscellaneous
Overstained preparationDestain with 95% methanol and restain
Refractile artifact on RBC with Diff-Quik stainChange the fixative


*Reference: Cowell & Tyler (1992), Cytology and Haematology of the Horse.

Cheers
Douglas