Sunday, 9 December 2007

MMIC - Follow up on Specimen 3.

Specimen 3:
Preliminary Information:

Patient: Maisy Hong (67 years)
Sample: Urine
Symptoms: Fever, chills, bladder distension; on indwelling catheter
Bladder distension: Inability to urinate. May be due to obstruction.
Indwelling catheter: prevent catheter from being pulled out of the bladder

Diagnosis: Urinary Track Infection

From week 1:

Possible causative agents: catheter- associated UTI

Most probable agents: according to incidence seen in UTI patients
1) Escherichia coli – 53-72%
2) Klebsiella spp. (pneumoniae) – 6-12%
3) Proteus spp. (mirabilis) – 4-6%
4) Enterococcus spp.(faecalis) – 2-12%


Additional suspected agents:
5) Pseudomonas spp. (aeruginosa)
6) Enterobacter spp. (aerogenes)
7) Serratia spp. (marcescens)
8) Candida spp. (albicans): may cause UTI, associated with use of catheter

Note: Chlamydia and Mycoplasma were excluded as these are typically sexually transmitted, highly unlikely to be the cause in a 67 year old woman with a catheter

Preliminary tests:
Specimen: Urine (dried on slide)

Stains:
Fungal stain
* Periodic acid-Schiff (PAS) can be used to detect if causative agent is fungi.
Gram stain
*Used to detect if bacteria is
- gram negative
- Escherichia coli (rod)
- Proteus Species (rod)
- Enterobacter Species (bacilli)
- Klebsiella Species (bacilli)
- Pseudomonas Species (bacilli)
*gram positive
- Enterococcus Species (cocci)

After the above test have been done, it would be clearer as to whether it is a fungi or bacteria. If it is a bacteria, it would have been narrowed down to gram type. Further screening as shown below for each individual suspected agent will then be performed if agent falls under the initial testing results.

Individual tests:

Escherichia coli
Morphology (microscopy): Gram negative bacilli, singly.
Pathogenesis: Adherence factor P fimbria binds specific galatose disaccharide found on uroepithelial cells surfaces. Siderophores produce cytotoxic alpha and beta hemolysins for lysis of urinary track cells for iron acquisition.
Key Characteristics: Rapidly ferment lactose, positive indole test, positive B-glucaronidase (using substrate MUG), facultative anaerobic (mixed-acid fermentation)
Tests:
1) EMB (Eosin Methylene Blue). Detect lactose fermenting colonies. Contains eosin and methylene blue.
· Black colonies with green sheen (positive, only E.coli can produce this colour due to acid production)
2) MacConkey Agar. Contains bile salts, crystal violet dye (to inhibit Gram-positive bacteria), neutral red dye (which stains microbes fermenting lactose).
· Red colonies (positive)
3) Indole test. Measures the ability of the microorganism to degrade tryptophan into indole, ammonia and pyruvic acid. Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48 hours prior to test. Following incubation, add 5 drops of Kovac's reagent.
· Red or red-violet color (positive)
4) Methyl Red (MR).
· Red (positive)
5) Citrate test. Utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source
· Green (negative)
6) MUG (on nutrient agar). Nutrient Agar with methylumbelliferyl-ß-D glucuronide (MUG) detects Escherichia coli (E.coli).
· Colonies with bright blue fluorescence
7) Blood agar plates (β-hemolytic)
· Hemolysis (Positive)
8) Urease Test
· Orange (Negative).
9) Triple Sugar Iron (TSI). Contains phenol red, high concentrations of lactose and sucrose, and a low concentration of glucose as well as sodium thiosulfate and ferric citrate . Provides aerobic and anaerobic conditions. Detects lactose fermentative bacteria – any bacteria that can ferment any of the three sugars.
· Red agar turns yellowish (positive). Acidic slant & butt (lactose and glucose fermenter)
· Stormy fermentation – production of hydrogen gas
10) Antibiotic Susceptibility :
· Amoxicillin, cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin and the aminoglycosides
11) Serology. Detection of O & H antigens (ELISA)
· Slide agglutination – Positive
· Tube agglutination - Positive
Biochemical summary:
IMViC
++--


Klebsiella spp. (pneumoniae)
Morphology (microscopy): Encapsulated gram negative rod (pH 5, acidic) may be cocci in more alkaline conditions (pH 7)
Pathogenesis: Type 1 fimbriae consisting of building block protein (FimA) extend beyond the capsular matrix and mediate adhesion to host epithelial cells.
Capsular polysaccharide matrix encases cell surface to provide resistance against host defence mechanisms.
Key characteristics: Negative indole test. Positive Voges Proskauer (Vi). Lactose fermenting, facultative anaerobic.
Tests:
1) MacConkey Agar.
· Red colonies (positive)
2)Triple Sugar Iron (TSI).
· Red agar remains red (negative). Alkaline slant & butt. (non-fermenter)
3) Indole test.
· Yellow (negative)
4) Voges-Proskauer (VP) test. Determines if neutral products such as ethyl alcohol, acetoin and butanediol are formed
· Pink-burgundy color (positive), 30mins to develop colour
5) Methyl Red (MR) test:
· Yellow (negative)
6) Citrate test. Utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source
· Prussian blue color (positive)
7) Urease Test.
· Orange (Negative).
8) Antibiotic Susceptibility :
· Third generation cephalosporin
9) Serology. Capsular serotyping
· K antigens
Biochemical summary:
IMViC
--++


Proteus spp. (mirabilis)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Mostly positive indole test, except P. mirabilis. Deaminate phenylalaine. Grow on potassium cyanide medium. Ferment xylose. Motile, swarming effect on agar plates. urease-positive. Facultative anaerobe.
Tests:
1) Oxidase test
· Negative
2) Triple Sugar Iron (TSI).
· Black precipitation (H2S production), red slant (alkaline). Glucose fermenter, non-lactose fermenter.
3) Indole Test.
· Most strains positive (red), mirabilis negative (yellow).
4) Methyl Red Test
· Red (positive)
5) Voges-Proskauer (VP) test.
· Negative.
6) Citrate test.
· Prussian blue color (positive)
7) Urease Test.
· Pink (Positive).
8) MacConkey Agar
· Light/colourless colonies (negative)
9) OILM medium. Test for ornithine decarboxylase utilization.
· Green/blue upper portion (Positive).
10) Blood agar plates
· Swarming observed
11) Antibiotic Susceptibility
· Penicillin derivatives, cephalosporins, quinolones, aminoglucosides
Biochemical summary:
IMViC
-+-+


Enterococcus spp.(faecalis)
Morphology (microscopy): Gram positive cocci. (diplococci)
Key Characteristics: catalase negative, colonies appear grey. Group D streptococci. gamma hemolytic (no hemolysis)
Tests:
1) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Negative
2) Blood plate agar
· No hemolysis
3) Bile-Esculin Hydrolysis Test. Determine the ability of an organism to hydrolyze the glycoside esculin to esculatin and glucose in the presence of bile (10 - 40%).
· Black to dark brown slant (Positive)
4) 6.5% salt broth. Test salt tolerance.
· Positive, growth.
5) PYR (pyroglutamyl aminopeptidase) disc
· Positive
6) Xylose fermentation test. performed with D-xylose tablets.
· Positive
7) MacConkey Agar. Without crystal violet for inhibition of gram positive bacteria.
· Pink (positive)
8) Serology. Western blot (IgG from patient’s sera). ELISA.
· Polysaccharide antigen extracted from bacterial cell walls detected
9) Antibiotic Susceptibility
· Quinupristin, ampicillin and vancomycin


Pseudomonas spp. (aeruginosa)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Aerobic (classified facultative anaerobe), rod-shaped bacterium with unipolar motility (due to single polar flagellum), opportunistic pathogen (seen in immunocompromised individuals). Pearlescent appearance and grape-like odor
Tests:
1) MacConkey Agar
· Colourless colonies - Negative (non glucose fermenter)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Blood plate agar
· swarming
4) Oxidase test
· Positive
5) Urease Test
· Positive
6) Indole Test
· Negative
7) Methyl Red
· Negative
8) Voges Proskauer Test
· Negative
9) Citrate test.
. Prussian blue color (positive)
10) TSI
· red slant (alkaline), red butt (alkaline) . Non fermenter of glucose
11) Antibiotic Susceptibility
· Aminoglycosides, Quinolones, Cephalosporins
12) Serology. Detect A, B , O antigens on cell wall
· Slide agglutination – Positive
· Tube agglutination - Positive
Biochemical summary:
IMViC
---+


Enterobacter spp. (aerogenes)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Facultative anaerobe, oxidase negative, catalase positive. Typically caused by catheter insertions
Tests:
1) MacConkey Agar
· Pink colonies - Positive, may be weak (glucose fermenter)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Oxidase test
· Negative
4) Urease Test
· Variable
5) Indole Test
· Negative
6) Methyl Red
· Negative
7) Voges Proskauer Test
· Positive
8) Citrate test.
· Prussian blue color (positive)
9) TSI
· red slant (alkaline), yellow butt (acidic) . Fermenter of glucose
10) Antibiotic Susceptibility
. Ciprofloxacin, Tazobactem
Biochemical summary:
IMViC
--++


Serratia spp. (marcescens)
Morphology (microscopy): Gram negative bacilli.
Key Characteristics: Produces lactose-fermenting colonies on differential agar, but is a late lactose fermenter may seem like a negative reaction (considered negative). Forms red-pigmented colonies. Able to perform casein hydrolysis and degrates tryptophan and citrate.
Tests:
1) MacConkey Agar
· Red colonies. Negative (initial)
2) Catalase test. Identifies organisms which produce the catalase enzyme; this enzyme converts hydrogen peroxide to water and oxygen gas
· Positive
3) Oxidase test
· Negative
4) Urease Test
· Positive
6) Indole Test
· Negative
7) Methyl Red
· Negative
8) Voges Proskauer Test
· Positive
9) Citrate test.
· Prussian blue color (positive)
10) TSI
· red slant (alkaline), yellow butt (acidic) . Fermenter of glucose
11) Antibiotic Susceptibility
. Aminoglycoside , Amikacin, Gentamicin, Tobramycin and Quinolones
Biochemical summary:
IMViC
--++



Candida spp. (albicans)
Morphology (microscopy): chlamydospores.
Pathogenesis: Produces extracellular proteinases from SAP genes, phospholipases B enzymes and lipases that are secreted for host infection.

Tests:
1) Southern Blot. Detect SAP genes using SAP1 probe.
· Positive.
2) Serology. Detect cell wall antigens - ELISA (CWP, PPM, CW)
· Positive.
3) Oxoid OBIS albicans Test. Rapid card-based chromogenic test that detects the presence of two enzymes specific to C. albicans and C. dubliniensis: b-galactosaminidase and L-proline aminopeptidase
· Magenta colour (Positive).



References:
textbookofbacteriology.net > e.coli.html
http://www.cpg-biotech.com/ > userguides > tech1 > p35681
http://www.mc.maricopa.edu/ > johnson > labtools > Dbiochem > imvic.html
iai.asm.org > cgi > content > abstract > 73 > 8 > 4626
aem.asm.org > cgi > reprint > 28 > 4 > 534.pdf
http://www.blackwellpublishing.com/ > eccmid15 > abstract.asp?id=36567
http://www.clevelandchiropractic.edu/ > ClassFiles-LA > Microbiology > entero.htm
www2.austin.cc.tx.us > microbugz > html > catalase_test.html

Cheers,
Debra, TG02

Case 5 follow up

WFH's case of wound infection.

Revised list of suspected microorganisms:
1. S. aureus
2. S. pyrogenes
3. Clostridium perfringens
4. Clostridium botulinum
5. Pseudomonas aeruginosa
6. S. faecalis (enterococcus)
7.Proteus mirabilis

Microorganism

Key characteristics

Lab diagnosis

S. aureus

Produce coagulase enzyme

Coagulase positive (agglutination)

Produce catalase

Catalase positive [Frothing when react with H2O2 ]

Ferments mannitol

Yellow colonies on mannitol salt agar

Lyses rbc

B haemolysis on BA

B lactamase

B lactam resistant. Sensitivity to naficin, methicillin, vancomycin


Gram positive (purple) cocci, “grape-like”

S. pyogenes

Lyses rbc

B haemolysis on BA

Does not produce catalase

Catalase negative


Bacitracin sensitive


Hippurate and cAMP negative


Gram positive cocci in chains

C. perfringens

Double zone of haemolysis

B haemolysis on BA

Alpha lecithinase

Lecithinase positive on egg yolk agar


Gram positive, large pink rods


Proteolysis on CM (meat turn black)

C. botulinum

Motile

Swarming effect on agar

Lipase positive


Lecithinase and protease neg

Meat at the bottom of CM remain brown


Gram positive, oval and subterminal spores, bacilli

P. aeruginosa

Non fermentative

Pink colonies on macconkey agar

Oxidase positive


Pyocyanin pigment

Blue on pseudomonas agar P

Pyoverdin pigment

Fluorescein on Pseudomonas agar F

Utilizes citrate

Citrate + (green to blue on citrate agar slant)


Urease+/-

Unable to hydrolyse and deaminate tryptophan

Indole -

Does not utilize carbohydrates

TSI-

S. faecalis

Non haemolytic

Gamma haemolysis on BA

Tolerant to 6.5% NaCl

Growth on mannitol salt agar

Resistant to bile


Hydrolyses esculin

Blackening of esculin agar

Does not produce B lactamase

B lactam sensitive


Gram positive cocci in chains

P. mirabilis

Unable to metabolise lactose

Pink colonies on MAC

Distinct ordor


Utilises urea and citrate

Citrate +

H2S production

Black precipitate on TSI

Unable to hydrolyse and deaminate tryptophan

Indole -


Nitrogen reductase -

Mixed acid fermentation

Methyl red +

Unable to ferment glucose

Voges- Proskauer -

Produce catalase

Catalase +


Oxidase -


Phenylalanine deaminase +




Yeng Ting
Tg 02

MMIC blog 2 - case 4

Probable microorganisms:

Streptococcus pneumoniae
- gram-positive
- alpha-haemolytic diplococcus bacterium
- optochin sensitive

Pathogenesis:
- produce IgA protease that enhances organism’s ability to colonize the mucosa of the upper respiratory tract. It multiply in tissue and causes inflammation.

Lab investigation:
- Gram stain
- Culture on blood agar plates
- catalase negative
- antibiotic susceptibility – susceptible to penicillins and erythromycin.

Moraxella catarrhalis
- gram-negative
- aerobic
- diplococcus
- produce beta-lactamases

Pathogenesis:
- cause respiratory tract-associated infection in humans

Lab investigation:
- Gram stain
- oxidase negative
- antibiotic susceptibility – susceptible to cephalosporins, resistant to penicillin

Haemophilus influenzae
- gram-negative
- coccobacilli
- non-motile
- generally aerobic, but can grow as a facultative anaerobe.

Pathogenesis:
- Their capsule allows them to resist phagocytosis and complement-mediated lysis in the non-immune host. Unencapsulated strains are less invasive, but they are able to induce an inflammatory response that causes disease.

Lab investigations:
- Gram stain
- culture on blood agar
- catalase positive
- oxidase positive

Mycoplasma pneumoniae
- causes infectious bronchitis

Lab investigations:
- lack of bacteria in a gram stained sputum sample
- lack of growth on blood agar
- positive blood test for cold hemagglutinins.
- Antibiotic susceptibility – susceptible to erythromycin

Chlamydia pneumoniae
- causes infectious bronchitis

Lab investigations:
- Giemsa stain
- Serologic test for antibody in patient’s serum.
- Antibiotic susceptibility – susceptible to tetracycline such as doxycycline

Bordetella pertussis
- gram-negative
- coccobacillus
- aerobic
- non-motile

Lab investigation:
- Culture on Bordet-Gengou agar plate with added cephalosporin select for the organism
- Oxidase positive
- urease negative
- nitrate negative
- citrate negative
- Antibiotic susceptibility – susceptible to erythromycin

Klebsiella pneumoniae
- gram-negative
- facultative anaerobic
- non-motile
- lactose fermenting

Lab investigations:
- Culture – MacConkey’s agar with lactose fermenting colonies.
- oxidase negative
- TSI: slant & butt yellow (acidic)
- H2S negative
- indole negative
- methyl-red negative
- Voges-Proskauer positive
- citrate positive
- urease positive
- Antibiotic susceptibility – susceptible to cephalosporin, resistant to penicillin

References:
http://www.merck.com> Lung and Airway Disorders> Bronchitis
http://www.wikipedia.org> Streptoccocus pneumoniae
http://www.wikipedia.org> Moraxella catarrhalis
http://www.wikipedia.org> Haemophilus influenzae

eunice
tg02

Saturday, 8 December 2007

Cheng Hong: MMic PBL Kuan Siew Yan follow up

There are some other organisms and viruses that are able to cause diarrhea like:

§
Clostridium perfringens
§
Staphylococcus aureus
§ Rotavirus
§ Norovirus
§ Vibrio cholerae



Clostridium perfringens
Characteristics: Rigid, thick walled cell, gram positive, spore forming anaerobic rods

Pathogenesis: Causes gas gangrene and food poisoning. C. perfringens is a normal flora in the colon but not found in the small bowels. If it is in the small bowels, the enterotoxin (super antigen on the C. perfringens) will cause diarrhea.

Why is not likely in this case: The diarrhea will only last for 24hrs and the disease will be resolved after 24hrs. This bacteria do not cause enterocolitis.


Staphylococcus aureus
Characteristics: Rigid, thick walled cell, gram positive cocci

Pathogenesis: Causes food poisoning. The enterotoxin causes food poisoning (vomiting/ watery, non-bloody diarrhea). The enterotoxin acts like a super antigen and stimulate the release of interleukin-1 and 2. May be transmitted through improperly cooked food as S. aureus is quite heat resistant.

Biochemical features: Coagulase positive, positive latex test, plate on Mannitol salt agar which acts as a selective medium and differential medium

Others: Smears will show gram positive cocci in clusters (purple).


Rotavirus
Characteristics:Reovirus family, non-enveloped, ssRNA virus, Icosahedral capsid

Pathogenesis: Causes diarrhea in young children, transmitted through fecal oral route. The virus multiplies in the small intestine and causing salt, glucose, water to be loss through diarrhea.

Diagnosis: Using ELISA techniques/ radioimmunology/ Rapid testing kits


Why it is not likely in this case: As Rotavirus infection mostly occurs in babies and very young children. Adults rarely will be infected as at the age of 6, children will have antibodies against at least 1 serotype of rotavirus.


Norovirus
Characteristics: Calicivirus family, non-enveloped, ssRNA virus, Icosahedral capsid

Pathogenesis: Transmitted through fecal oral route, or ingesting contaminated seafood/water. Virulent (low infectious dose). May cause vomiting, fever, diarrhea.

Diagnosis: PCR method or ELISA method



Vibrio cholerae
Characteristics: Rigid, thick walled cell, Gram negative, Facultative curved rods (comma shape)

Pathogenesis: Transmitted through fecal contamination in water and food, found in shellfish/ oysters. It causes watery diarrhea by causing the cells in the gut to lose water and ion. It does not cause bloody diarrhea or abdominal pain.

Biochemical features: oxidase positive, acid slant and acid butt, but no gas or H2S on TSI. Can be confirmed usingagglutination test (polyvalent O1 or non-O1 antiserum.

Please click the link below to view the biochemical test. (Maximise the table for better viewing)

http://www.geocities.com/alyssialam/biochemicalTest.gif

References from:Review of Medical Microbiology and Immunology(9th edition). WARREN LEVINSON
:Color Atlas of Medical Bacteriology. ASM PRESS

MMIC Blog 2 - Case 6

[A follow-up on my patient, Ong Fei Fei's investigation]


Laboratory Diagnosis

Since the patient has been previously diagnosed with UTI, the microbe could have move down and infect the vagina as well, hence urethra microbes are considered in this case too, for examples:

E. coli
Proteus mirabilis
S. saprophyticus
Group B streptococci (Alpha & non-hemolytic)
Coagulase-negative Staphylococci (S. aureus)
Klebsiella species
Proteus species
Pseudomonas aeruginosa
Enterobacteraceae

(Follow how UTI microbes are identify in other cases - see debra's and/or elaine's)


As for more vaginal-specific microbes, they are the followings:


Trichomonas Vaginalis


  • Wet mount preparation then Direct microscopy


  • Motile characteristic


  • Greater sensitivity: Fixed stained preparation (Giemsa/Papanicolaou) then Direct Immunofluorescence


  • Culture (Diamond’s medium) then incubate at 37oC à microscopy


  • Antibody detection techniques: high in false positive and false negative results


  • Enzyme immunoassay for detection of T. vaginalis antigen


Candida Albicans




  • Gram’s stain then Direct Microscopy


  • Sabouraud’s agar then Incubate 24-48 hours. white, butyrous colonies observed if positive
Bacterial Vaginosis (anaerobic/non-specific)





  • Clue cells (Vaginal epithelial cells with edges darkened by presence of numerous small bacteria adhering to their surface)


  • pH (>4.5)


  • Amine test: add few drops of KOH and presence of amines gives a fishy smell


  • Gram’s stain


  • Culture: for Gardnerella vaginalis and Mobiluncus species

1. Gardnerella vaginalis

Aerobic
Gram-variable
Bacillus
Slow growing
Non-motile
Catalase and oxidase negative
Beta-hemolysis on human blood agar but not on sheep blood agar
Selective blood agar: add gentamicin, nalidixic acid and amphotericin B
Hippurate hydrolysis: positive
Starch fermentation: positive
Metronizadole 50ug disc: sensitive
Sulphonamide 1000ug dsc: resistant


2. Mobiluncus species

Anaerobic
Gram-variable
Bacillus
Divided into: M. curtisii and M. mulieris
Fastidious; slow growing
Typically motile, catalase, oxidase, indole negative
Clear, colourless colonies (2mm) after 5 days incubation
Gas-liquid chromatography distinguish species
Commercial kit: detect enzyme activity (praline aminopeptidase and alpha-D-glucosidase)


3. Neisseria Gonorrhoea

Die readily outside human body
Fastidious
Use of rich media supplemented with yeast extract or iso-viatalex and blood (Chocolate agar)
Antibiotic inhibitors: vancomycin, nystatin, colistin, trimethoprim
Direct Microscopy (less sensitive for women – 50%)
Culture
Other technique: Direct immunofluorescence


4. Chlamydia trachomatis

[Direct examination of smear with fluorescein-conjugated monoclonal antibodies(Ab) (use of commercial kits)]

Roll specimen gently on slide
Fix with methanol for 4 min
2 Ab (one directed to the outer membrane – species specific, one specific to the genus lipopolysaccharide)
Subjective/ false positive
Suitable for small sample numbers and rapid screening


[Culture]

Uses McCoy’s cells treated with cyclohexamide
Centrifuged and incubate for 72 hours
Detection by Giemsa stain/iodine/fluorescein-labeled monoclonal Ab
Sensitivity: ~80%


[Enzyme-linked immunoabsorbent assays (ELISA)]

Uses polyclonal and monoclonal antibodies against lipopolysaccharide (Antigen detection)
Sensitivity: ~97%; Specificity: ~92.5% (improves by blocking tests)
False positive due to cross reactivity with other bacteria


[Nucleic acid probes]

DNA hybridization
Highly specific but lack sensitivity


[Serology]

Complement fixation tests are insensitive; difficult to differentiate the serotypes
Micro-immunofluorescence test detect specific IgG/IgM; difficult in sexually active populations



Summary Diagrams






























Other Investigation required


Urinalysis - for screening purpose; inexpensive and easy to perform (previously confirmed UTI)
Urine culture - for accurate diagnosis of infection to determine complications, such as antimicrobial susceptibility of infecting bacteria (previously confirmed UTI)


References

http://www.escriber.com/ > TrendsInUGSH > Features

http://www.cfps.org.sg/ > sfp > 23 > 232> articles > e232136.html

http://cks.library.nhs.uk/ > uti_lower_women > in_depth > goals_and_outcome_measures

http://www.merck.com/ > mmpe > sec17 > ch231 > ch231b.html

http://www.aafp.org/ > afp > 20020415 > 1589.pdf

http://books.mcgraw-hill.com/ medical > firstaidfortheboards > pdf > 0071443363 > 0071443363_282.pdf

http://classes.kumc.edu/ son > nrsg835 > gyninfect.htm



Posted by: Pei Shan, TG02

Friday, 7 December 2007

Elaine Blog2: case 1 Khong Fay seah

The entrobacteriaceae are heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of humans and animals. The family includes: Escherichia, shigella, salmonella, enterobacter, klebsiella, serratia.

E.coli form circular, convex, smooth colonies with distinct edges. Klebsiella colonies are large and very mucoid and tend to coalesce with prolonged incubation.
Salmonella and shigellae produce colonies similar to E.coli but they do not ferment lactose. However, salmonella and shigellae do not appear in the urinary tract.

E.coli is the most common cause of UTI and accounts for approximately 90% of first urinary tract infections in young women. The symptoms and signs include urinary frequency, dysuria, hematuria, and pyuria. However, none of these symptoms or signs is specific for E.coli.


procedure:


  • Urine collected from clean-catch mid-stream or one obtained by bladder catherterization or suprapubic.

  • common causative: E.coli; Enterobacteriaceae; other gram-negactive rods

  • usual microscope: gram-negative rods seen on stained smear of uncentrifuged urine indicate more than 105/mL

  • culture on blood agar and macConkey

  • Comments: gray colonies that are B-hemolytic and give a positive spot indole test are usually E.coli; other require further biochemicsl tests

E.coli--> colonies show metallis on EMB agar


Klebsiella pneumoniae--> has large mucoid capsule and hence viscous colonies


Proteus mirabilis--> motility causes "swarming" on agar; produce urease


Pseudomonas aeruginosa--> blue-green pigment and fruity odor produced; causes nonsocomial infections and often drug-resistant


If unknown-form of colonies are observed (other than E.coli) , biochemical tests should be done to differentiate each micro-organism from each other.


Diagnosis: Biochemical Reaction:


IMViC (Indole Methy Re€d Voges-Proskauer simmons's Citrate) plus lactose fermentation & TSI


enterobacteriaceae



  1. E.coli ++--+ acid slant/acid butt with gas production

  2. Pseudomonas Aeruginosa -+--- alkaline slant/alkaline butt plus positive oxidation fermentation

  3. Klebsiella Pneumoniae +-+++ acid slant/acid butt with gas production

  4. Proteus Mirabilis -+++- alkaline slant/acid butt with H2S production

Other possible bacteria and tests required:


5. Enterococcus Faecalis



  • growth in the presence of bile, hydrolyze esculin

  • Alpha-hemolytic

  • growth in 6.5% NaCl, PYR-positive

6. Staphylococcus Saprophyticus



  • -ve mannitol

  • -ve coagulase

  • -ve Novobiocin sensitivity

  • -ve alpha toxin

7. Neisseria Gonorrhoeae



  • +ve glucose

  • -ve maltose

  • -ve Lactose

  • -ve Sucrose

8. Mycoplasma Genitalium



  • culture is difficult.

  • Data obtained from PCR, molecular probes, serologic tests

9. Ureaplasma Urealyticum



  • It required 10% urea for growth


Antibiotic Susceptibility Testing:


Escherichia coli (gram -ve rods enteric and related organisms)
drug choice: Cefotaxime, Fluoroquinolones, nitrofurantoin

Pseudomonas aeruginosa (gram -ve rods aerobic)
drug choice: Aminoglycoside, pencillin

klebsiella pneumoniae (gram -ve rods enteric and related organisms )
drug choice: A cephalosporin

proteus mirabilis (gram -ve rods enteric and related organisms)
drug choice: Ampicillin


Staphylococcus saprophyticus (gram +ve cocci)
drug choice: penicillin, tetracycline, vancomycin, gentamicin, rifampin

Neisseria gonorrhoeae (gram -ve cocci)
drug choice: ceftriaxone, ciprofloxacin, gatifloxacin,

Enterococcus faecalis (Pos cocci)
drug choice: Ampicillin, gentamicin

Mycoplasma genitalium (wall-less cells)
drug choice: erythromycin (ERY), clarithromycin (CLR), ciprofloxacin hydrochloride (CIP)

Ureaplasma urealyticum
drug choice: ciprofloxacin and ofloxacin, tetracycline and doxycycline, roxithromycin, erythromycin

Monday, 3 December 2007

MMIC BLOG 1 - Case 6

Preliminary Information

Name: Ong Fei Fei
Sex: Female
Age: 37
Clinical Diagnosis: Urinary Tract Infection (UTI)
Symptoms: Fever, pain during urination, virginal discharge
Anitbiotic Treatment: none
Specimen: Vaginal discharge


Introduction

[UTI]
  • More common in female than male
  • Infection occurs less frequently in men because the urethra is much longer and the distance between the anus and urethral meatus is greater than in women
  • Prostatic secretions also confer a degree of antibacterial activity
  • A single episode of UTI in women is usually uncomplicated
  • UTI infection is limited to the bladder - the responsible organisms are usually gram negative coliforms
  • Ascending infection from the bladder can cause acute pyelonephritis which typically presents with loin pain, fever and chills, costovertebral angle tenderness, nausea and vomiting
  • A woman with UTI symptoms without significant bacteriuria finding on culture = acute uretheral syndrome (in 30% cases; 70% bacterial infections)
  • Patients with acute uretheral syndrome can be either with pyuria on urinalysis (70%) or without (30%)
  • Those with pyuria have true microbial infection usually with chlamydia; those without pyuria have no known microbial cause and the dysuria (a burning sensation in the urethra during voiding) and frequency may be related to irritation from mechanical trauma
  • Mostly caused by Commensal colonic gram-negative aerobic bacteria (> 75%)E.g. E. coli strains (with specific attachment factors for transitional epithelium of the bladder and ureters), other enterobacteria, especially Klebsiella, Proteus mirabilis, and Pseudomonas aeruginosa.
  • Enterococci (group D streptococci) and coagulase-negative staphylococci (eg, Staphylococcus saprophyticus) are the most frequently implicated gram-positive organisms.
  • Infection Localization - Vaginitis is often distinguished by the presence of vaginal discharge, vaginal odor, and dyspareunia

[Vaginal Discharge]
  • It is a secretion produced from glands in the vaginal and the cervical lining
  • All women have a little discharge starting approximately a year before puberty and ending after the menopause
  • A normal physiological discharge is usually clear, creamy or very slightly yellow
  • Abnormalcy when there is suddenly a great amount of discharge or the color is brownish, reddish or greenish or it becomes smelly.
  • Pathogens implicated are bacterial, fungal and protozoan
  • May be associated with Sexually Transmitted Disease (STDs); due to hypoestrogenism

Etiology

Trichomonas Vaginalis
  • Transmission: STD
  • Symptoms: Frothy green/yellow discharge, pruritus, urinary symptoms. May be asymptomatic (50%)
  • Protozoa found only in tropozoite stage, with 4 flagella
  • May be seen in urine
  • Offensive odour
  • Vagina may be inflamed (Complain of Vaginal/Vulval pruritis)
  • May have cervical erosions
Candida Albicans

  • A type of fungal (yeast) infection
  • Transmission: Increased with pregnancy, Diabetes Mellitus & antibiotics.
  • Symptoms: Discharge is white, resembles milk curds; Severe itching, dysuria, dyspareunia.

Bacterial Vaginosis (anaerobic/non-specific)

(Rarely presented with dysuria or dyspareunia)

1. Gardnerella vaginalis
Transmission: unknown; may be STD
Symptoms: Thin, watery, yellow-gray discharge with "fishy" odor

2. Mobiluncus species

3. Gonorrhoea
Transmission: STD (More easily transmitted from an infected man to womon ~90%)
Symptoms: Purulent vaginal discharge, dysuria, urinary frequency, inflammation, vulvar swelling and the cervix may be eroded. Most women are asymptomatic.

4. Chlamydia trachomatis (C. trachomatis, C. psittaci, C. pneumoniae)
Transmission: commonly STD
Symptoms (mild): May have thin/purulent discharge, urinary burning and frequency, lower abdominal pain and friable cervix. Women usually asymptomatic.


Possible Interpretation
Vaginal Discharge - suggests Vaginitis or urethritis (e.g. Sexually transmitted Disease (STD), candidiasis) due to hypoestrogenism: e.g. T. vaginalis or N. gonorrhoeae.


Investigation required
  • Urinalysis - for screening; inexpensive, easy to perform
  • Urine culture - for accurate diagnosis of infection to determine complications, such as antimicrobial susceptibility of infecting bacteria
  • Vaginal and urethral discharge - wet-mount preparation to detect Trichomonas vaginalis and Candida species
  • Gramstaining - detect Neisseria gonorrhoeae

References:

http://www.escriber.com/ > TrendsInUGSH > Features
http://www.cfps.org.sg/ > sfp > 23 > 232> articles > e232136.html
http://cks.library.nhs.uk/ > uti_lower_women > in_depth > goals_and_outcome_measures
http://www.merck.com/ > mmpe > sec17 > ch231 > ch231b.html
http://www.aafp.org/ > afp > 20020415 > 1589.pdf

Posted by: Pei Shan, TG02

MMIC PBL - Case 3

Specimen 3:
Preliminary Information:
Patient: Maisy Hong (67 years)
Sample: Urine
Symptoms: Fever, chills, bladder distension; on indwelling catheter
Bladder distension: Inability to urinate. May be due to obstruction.
Indwelling catheter: prevent catheter from being pulled out of the bladder
Diagnosis: Urinary Track Infection

- Women are more prone to UTI as the urethra is much shorter and closer to the anus than in males. Also, they lack the bacteriostatic properties of prostatic secretions.
- Use of urinary catheters (foreign body) in elderly may result in an increased risk of urinary tract infection.

Possible causative agents:

1. Bacteria: Causes up to 85% of all UTI cases.
(a) Escherichia coli: Most common causative agent of UTI, typically from bowels (colon).

(b) Enterococcus Species: Originate from colon.
E. faecalis

(c) Klebsiella Species: Opportunistic pathogens that causes nosocomial infections
K. pneumoniae

(d) Enterobacter Species: Opportunistic pathogen; Present in large intestine but also present in soil and water.
E. aerogenes
E. cloacae
E. taylorae

(e) Proteus Species: Have urease activity that raises urinary pH. Causes stone formation.
P. mirabilis

(f) Pseudomonas Species: Causes UTI primarily in patients with lowered host defences
P. aeruginosa

2. Fungi: Account for 40% of nosocomial UTI cases.

(a) Candida Species: may cause UTI in diabetic patients
C. albicans - causes vaginitis and chronic mucocutaneous candidiasis


As patient has a catheter, it is likely associated with contributing to UTI.
In this case, most incidences are caused by bacterial infection/colonization of the catheter, hence bacteria are suspected to be the cause.
Fungal stain may be used to rule out fungal infection in this case.

To narrow down type of bacteria, a gram stain should be done, to determine gram stain as well as morphology or bacteria:
Gram Stain:
Negative (pink): Escherichia coli (rod), Proteus Species (rod), Enterobacter Species (bacilli), Klebsiella Species (bacilli), Pseudomonas Species (bacilli).
Positive (purple): Enterococcus Species (cocci)

Gram negative strains are the most common causes; hence further biochemical test, urine cultures and antibiotic susceptibility must be employed to narrow down causative agent.

Most probable agents: according to incidence seen in UTI patients
Escherichia coli – 53-72%
Klebsiella pneumoniae – 6-12%
Proteus mirabilis – 4-6%
Enterococcus faecalis – 2-12%

References:
calder.med.miami.edu > pointis > indwelling.html
wrongdiagnosis.com > medical > bladder_distension.htm
kcom.edu > faculty > chamberlain > Website > lectures > lecture > uti.htm

Cheers,
Debra (TGo2)

MMIC - case 4

Name: Tong Wei Hong
Age: 68 yrs old
Sex: Male

Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis
Specimen: Sputum

Bronchitis is a respiratory disease in which the mucous membrane in the lungs' bronchial passages becomes inflamed. As the irritated membrane swells and grows thicker, it narrows or shuts off the tiny airways in the lungs, resulting in coughing spells accompanied by thick phlegm and breathlessness. There are two main types of bronchitis: acute and chronic.

- Several viruses cause bronchitis, including influenza A and B.

- A number of bacteria are known to cause bronchitis, such as Mycoplasma pneumoniae.

- Bronchitis also can occur when you inhale irritating fumes or dusts. Chemical solvents and smoke, including tobacco smoke, have been linked to acute bronchitis.

- People at increased risk both of getting bronchitis and of having more severe symptoms include the elderly, those with weakened immune systems, smokers, and anyone with repeated exposure to lung irritants.

Microorganisms commonly isolated from sputum include:
- Streptococcus pneumonia,
- Haemophilus influenzae
- Staphylococcus aureus
- Legionella pneumophila
- Mycoplasma pneumonia
- Klebsiella pneumoniae
- Pseudomonas aeruginosa
- Bordetella pertussis
- Escherichia coli

Cultures and tests are done on the sputum to help identify the bacteria that are causing an infection in the lungs or the airways (bronchi).
· Bacterial culture - gram stain or acid fast stain of the sputum done at the same time can help make the diagnosis.

· Fungal culture - The sputum sample is spread on special culture plates that will encourage the growth of mold and yeast. Different biochemical tests and stains are used to identify molds and yeast. Cultures for fungi may take several weeks.

· Viral culture - sputum is mixed with commercially-prepared animal cells in a test tube. Characteristic changes to the cells caused by the growing virus help identify the virus.

References
bronchitis>causes/">http://www.emedicinehealth.com>bronchitis>causes
http://www.lifesteps.com>Home > Encyclopedia > Encyclopedia Index S

Eunice
TG02

Sunday, 2 December 2007

dPBL MMIC:cheng hong (enterocolitis)

Name: Kuan Siew Yan / 29yrs/ Female

Symptom(s):Diarrhoea
Diagnosis: Enterocolitis


What is Enterocolitis?
Cause by organisms that invade the epithelial and the subepithelial tissue of the small and large intestine thus causing inflammation and diarrhea.

Possible Causative Agents
1.Salmonella species
2.Shigella species
3.Campylobacter jejuni
4.Escherichia coli O157

1. Salmonella
§ Characteristics: Rigid, thick walled cell, Gram negative, Facultative straight rods

§ Species: S.typhi, Salmonella choleraeuis, Salmonella enteritidis


§ Pathogenesis: Manifest as Enterocolitis (penetrates through mucosal cells into the lamina propria; need at least 100000 organisms to cause infection). Typhoid/enteric fevers or Septicemia.

§ Biochemical features: Non lactose fermenting colonies on MacConkey/ EMB agar
: Alkaline slant/ acidic butt, gas and H2S in butt in TSI
: Lysine, Aginine, Ornithine positive
: indole neg, methyl red pos, Voges-Proskauer neg, citrate neg


2. Shigella species

§ Charateristics: Rigid, thick walled cell, Gram negative, Facultative straight rods, non-motile

Species: 4 different genus groups (A,B,C,D)


Pathogenesis: virulent (only need 100 organism to cause the disease), transmitted through fecal-oral route, normally have bloody diarrhea

Biochemical features: non-lactose fermenting, no gas production from fermenting glucose, no H2S production, alkaline slant and acidic butt on TSI, indole neg, methyl red pos, vogues proskauer neg, citrate neg, lysine arginine ornithine neg

Other features: a methylene blue stain will differentiate if it is an invasive or toxin producing bacteria (neutrophils will show that it is an invasive infection; like Shigella, Salmonella, Campylobacter


3. Campylobacter jejuni
§ Characteristics: Rigid, thick walled cell, Gram negative, Facultative curved rods (S shape)

§ Species: C. jejuni, C. intestinalis


§ Pathogenesis: Transmitted through fecal oral route or food/water contaminated with Campylobacter, causes entrocolitis

§ Biochemical features: culture on blood plate containing antibiotics (vancomycin, trimethoprim, cephalothin, polymyxin, amphotericin B) and incubate at 42oC at 5%oxygen and 10% carbon dioxide, no H2S production on TSI, hippurate hydrolysis test pos for C. jejuni, susceptible to nalidixic


§ Others: hanging drop shows motility of Campylobacter


4. Escherichia coli O157
Characteristics: Rigid, thick walled cell, Gram negative, Facultative straight rods

Pathogenesis: Enterohemorrhagic infection due to ingesting undercookedmeat, the organism cause the disease by using its features (pili, capsule, endotoxin, 3 exotoxins)-results in watery/ bloody diarrhea


Biochemical features: Can be either lactose fermenting/ non lactose fermenting, appears green on EBM agar, produces indole from tryptophan, decarboxylates lysine, motile, O157:H7 does not ferment sorbitol, acidic slant acidic butt produces gas abd no H2S production

References from:Review of Medical Microbiology and Immunology(9th edition). WARREN LEVINSON

:Color Atlas of Medical Bacteriology. ASM PRESS

Cheng Hong

MMIC

Name: Khong Fay Seah
Age: 27 yrs old
Sex: Female

Complaints: Fever, chills, dysuria (painful urination)
Diagnosis: UTI
Specimen: Urine

Test ordered (Main Lab): Urine FEME (microscopy, dipstick)

Procedure:

Step 1:
A drop of uncentrifuged urine placed into a KOVA chamber, and examined with restricted light intensity under the high-dry objective of an ordinary clinical microscope can reveal leukocytes, epithelial cells, and bacteria.

Finding 105 organisms per millililiter can conclude UTI.
Results:
Normal hematocrit/HB
Elevated WBC (>18,000/uL)
Innumerable white blood cells
A few RBC
Numerous bacteria

Step2:
Positive Urine dipstick nitirite suggest strongly bacteria growth in the urine tract.

Step 3:
A gram-stained smear of uncentrifuged urine that shows gram-negative rods is diagnostic of UTI

Step 4: in the micro lab

· Culture urine using a bacteriologic loop calibrated to deliver 0.01 or 0.00mL to agar plates.
· Aerobic culture ( maconkey and blood agar plates) & incubated overnight @ 37 degree.

In active pyelonephritis, the number of bacteria in urine collected by uretheral catheter is relatively lo.

While accumulating in the bladder, bacteria multiply rapidly and soon reach >105/mL (not contamination). The presence of more than 105/mL of the same type of bacteria per milliliter, establish a diagnosis of active infection. E.coli might be causing bacteria.

If the growth of different types of bacteria fewer than 104/mL, suggest that organisms come from normal flora or contaminants.

This female patient with acute dysuria and UTI will have 102/mL-103/mL.

If the cultures are negative but clinical signs of UTI are present, ureteral obstruction, tuberculosis can be considered.

E.coli cause 80-90% of acute bacterial lower tract infection in young women. The patient with acute cystitis has negative urine cultures for bacteria. Other possible causing bacteria: Staphylococcus, Neisseria Gonorrhoeae, Klebsiella, Enterobacter.

If such infection is caused by E.coli, identification and susceptibility testing of bacteria are not necessary.

elaine

Wednesday, 28 November 2007

M.mic- Case on WFH

Case: Surgical wound infection
Specimen: Wound swab

There are 2 main types of culture for wound infection. For external wounds (on the surface of the skin), swabs are sent in dry tube and stuart transport medium. The dry swab is used for gram staining to prelimarily identify organisms present on the wound. We normally look out for gram positive or negative organisms, bacilli or cocci. Afterwhich, the swab sent in stuart transport medium is used for culturing on blood agar (BA), macconkey agar (MAC) and cooked meat agar (CM).

BA is used for general growth of organisms and identifying streptococcus strains based on the hemolytic activities (alpha, beta or gamma hemolysis).

MAC is used as an inhibitory and differential medium used to distinguish lactose-fermenting enteric gram-negative bacilli from nonfermenters.

CM is used for isolation of Clostridium and to evaluate proteolysis.


For internal wounds (below the skin surface area), swabs are sent in dry tube, PRAS transport medium and stuart transport medium. The procedures is the same as above for the dry and stuart swabs. For the PRAS swab, we use it to culture on another BA for incubation in CO2 and ANO2

Since a dry swab will be provided, the external culture will be done.

The procedure:

1. Culture on blood agar
2. Culture on Macconkey agar
3. Culture on cooked meat agar
4. Do a gram stain

Gram stain is done after plating because it will contaminate the swab. (the slide is not sterile)

The most common causative organisms isolated for wound infection are:

1. Styphylococcus aureus.
2.Streptococcus pyogenes
3.Clostridium
4. Pseudomonas
5. Enterococcus
6. Proteus mirabilis
7. Enterobacteriaceae


References


Lehmann (1998) Clinical Bacteriology. Saunders Manual of Clinical Laboratory science pages 589 to 667. WB Saunders, Philadelphia

SOP of attachment company.

Tuesday, 6 November 2007

Elaine"reverse camp test"

hey everyone,

W.R.T last post, I found a case study online about the last post on Acranobacterium hemolyticum


If you are free, please visit this website. It show clearly the symptoms and the stages of how a Medical Technologist will do if he/she suspect something.



To lizze, arcanobacterium hemolyticum is quite a difficult to culture out because it need a strictly anaerobe environmemt. If the colonies are too small, B-hemolytic ability is also difficult to observe. The med tech will normally re-isolate the microorganism on a new sheep blood plate because the arcanobacteria will not survive once it is out in the open-air.


This above pic(from the same website) show a clear reaction. It is quite difficult to get this. I don't even get to see a clear one like this.
Elaine

Friday, 26 October 2007

Time to clear all queries...... for cholesterol measurement!

Cheng Hong said:
Is there any other test that is done together with this test to confirm the results in case there is interferance due to bilirubin?

I say:
Pls note that a bilirubin of up to 20mg/dL do not interfere with the result. Anyway a single cholesterol measurement does not truly reflect a person's usual cholesterol concentration, it is highly recommended to perform the test twice on 2 separate occasions. The best is to encourage the participant to go to our Hospital Clinic for a thorough Liver Function check up (if the result seems weird) before a clinical diagnosis is finalised. Frankly speaking, we're trying to promote our health screen packages at the outreach program too.

Elaine said:
As you said, you went for outreach programme and did this cholesteral measurement. If the participant have the habit of eating vit. C pills everyday(is it the same as IV infusion?), does it means that he/she cannot do this test?

I say:
Since vitamin C pills are water soluble, they are flushed out of our immune system on a daily basis and do not have any significant impact on the measurement unless there is an over dosage jus before the test or having an IV infusion concurrently. This would then caused a falsely low cholesterol level.


Jue Xiu said:
hihiI just wonder as u mention the test will be affected by the 1.)IV infusion of Vitamin C2.)Elevated bilirubin (>171umol/L); Jaundice3.)Haematocrit > 55%4.)Methylaminoantiprine5.)Gentisic AcidWouldnt there be any discrepancy regarding the result? Or this test is specific for certain people due to the limitation?

I say:
helloo... I would say no, its not specific since it is used for point-of-care testing. However i would say yes for result discrepancy based on the degree of interference caused by what i've mentioned. Actually all tests have their limitations, so it's better for the particpants to have their blood serum tested too - sell health packages!

Vino said:
hey hey U mentioned abt detection reagent present in the yellow region of the test trip. wat is/ are the detection reagent?? that causes the reaction, colour change, to occur?

I say:
Sorry Vino, I could not find answer to your question as i believe the manufacturer is unwilling to reveal their secret reagent recipe. However, in parallel with the detection reagent from another brand, it was revealed that it contains microorganisms esterase and oxidase, 4-aminoantipyrine, horseradish peroxidase & substituted aniline derivatives. The reaction/colour change occurs due to liposis of the triglycerides and then measurement by photometry (refer to below, Cass's Question & my answer)


Charmaine said:
Just want to say, I learnt to use this as well!But I'm not measuring blood cholesterol level, instead I'm measuring blood glucose levels. XDCheers~Charmaine TanTG01

I say:
Oh... we too! We actually perform the finger-prick for the blood cholesterol 1st and squeeze another drop of blood for the glucose measurement as it takes a shorter time for the glucose device. While waiting for the 3 min to be up, I then measure the participant's blood pressure. Usually they would complain that their blood pressure is high due to the shock/fear that they develop for the finger-prick. All excuses.... I find it the same to measure immediately when they were jus made to sit down.

Avery said:
Hi Pei shan,i would to ask how do you select the depth of the Accu-Chek Safe-T-Pro Plus® lancet device?based on the finger thickness(hehe ^_^)?

I say:
Bingo! The lancet is usually set at a moderate depth which is sufficient to prick a person's finger. However if the person has thick skin especially the males, shift it to the deeper profile. So far that day, I only adjusted to the deepest twice, both times for men! It's better to adjust the depth than to be sorry and prick another time.

Cass said:
hi pei shan;)"The meter measures the intensity of the reaction colour based on reflectance photometry and calculates the concentration of the sample through a "lot-specific" algorithm (i.e. the data previously entered by mean of code strip), giving the cholesterol reading."what is reflectance photometry? intensity of the reaction.. meaning the darker the colour the higher the cholesterol level?

I say:
Helloooo... This is a very good question which I've lacked explaining. The reaction principle of Accutrend® Triglycerides is lipolysis of triglycerides to give free glycerol and fatty acids by the activity of a cholesterol esterase. The glycerol is phosphorylated by glycerol kinase, and the resulting glycerol phosphate is oxidized by a glycerol-phosphate oxidase. Molecular oxygen subsequently leads to the formation of hydrogen peroxide, whose oxidation equivalents are transferred by a peroxidase to an indicator, giving a blue-gray oxidation product. The intensity of the color is measured by reflectance photometry(American Association for Clinical Chemistry, Inc.) . The darker the colour, the higher the cholesterol level and the device converts this reading into a result for display.
For further reading, pls refer to http://www.clinchem.org/cgi/content/full/46/2/287

Pei Shan
TG02

Microbiology--- CAMP TEST (week 19)

hey to all!!!

I'm posting for week 19!!! TIme passed super fast...
I saw ying ying's posts abt CAMP test but what i'm abt to post is a bit different from her.

Here i go...

For those who had been to micro lab, you should pretty well known with GPB/GPC/GNB/GNC.
Here i'm going to introduce you to ARCANOBACTERIUM HAEMOLYTICUM.

CHARACTERISTICS:

--- gram postive rods with pointed ends

---catalase negative
---small colonies, may be smooth, mucoid, white, dry
---B-haemolytic
---It will not grow on MacConkey agar
--- Facultative anaerobe

Certain organism such as Group B streptoccui able to produce a factor that acts synergistically with B-hemolytic of S.aureus subspec. aureus on sheep blood. The purpose of test to determine the organism's ability to produce and elaborate CAMP factor. The production of a-hemolytic(a-toxin) by clostridium perfrigen also demostrate the synergistic phenomenon with CAMP factor. If Phospholipase D is produced by organism, the CAMP reaction would be inhibited.


Medium:
TSA with 5% sheep blood agar (BAP)


Procedure:
1) preheat 35'C. Ensure plates are dry.
2) Using inoculating needle, streak B-lysin substrate organism in a straight and across centre of BAP
3) Streak QC stain ( about 2-3cm) perpendicular to S.aureus inoculum without touching the Staphylococcui
4) Incubate CO2 35'c incubator for at least 24 hours
**Note: the arrow head means +ve CAMP results
The curve head means -ve CAMP results



MY senior said that normally she will do another REVERSE CAMP test(RCT) to confirm the results from CAMP test.


PRCT-- production of a distinct crescent hemolysis at junction between streptoccus agalatiae streak and test organism.

NPRCT-- No constriction of band of hemolysis






**Note: Postive reaction

Limitation:

--> size of zone is depend on the amount of bacterial product and its diffuse rate through the medium

--> inocula should be sufficient to produce confluent growth. The extent and intensity of lysis depends on the size of the staphylocuccus inoculum. If the inocula is too light, reaction would be weak.

--> If the inocula are not perpendicular, production of arrow heads will not appear. False Negative occur.







ELaIne

SEE ya soon!!!





Thursday, 25 October 2007

Research - Lab Techniques (week 18)

Hi all,

Previously I said I’d cover colony pcr and restriction digestion, but I believe those underlying principles have been covered in my first post, so it’s a bit redundant. Hence, I’ll be covering a bit on the yeast work for today.

First off, my protein interaction study is based on yeast. There are about 3 types of yeast 2 hybid assay. For my project, I’m using yeast 2 hybrid system 2. As I haven’t been exposed to the other 2 systems, I’m afraid I will not be able to cover anything on it.

Basically, the main principle of the yeast 2 hybrid is that the transcription factor in yeast, (GAL4) can be broken up into 2 pieces to form GAL4-BD (binding domain) and GAL4-AD (activation domain).
- GAL4-BD will then be subcloned into a vector for the bait gene, which is the gene of interest for study. The BD is the domain responsible for binding to the UAS (upstream activation site)
- GAL4-AD is subcloned into a vector for the prey cDNA library which is used for testing against the bait gene to see what interactions may take place. AD is the domain responsible for activation of transcription located downstream.

Hence, in event of a protein interaction between the bait and a particular prey, the 2 domains (GAL4-BD and AD) will be brought into close proximity, linking both the GAL4 UAS and transcriptional site in yeast, resulting in transcriptional activation of the beta/alpha-galactosidase, the reporter gene in yeast (MEL1, the reporter for producing alpha-galactosidase is naturally occurring in some strains of yeast, while LacZ is an E.coli enzyme that is cloned into the yeast genome).
Yeast strain that can metabolize alpha-galactosidase such as AH109, Y187 etc from clontech can metabolise x-alpha-gal (a substrate) directly as alpha-galactosidase is secreted (naturally occurring in these yeast strains); colonies can be plated on x-alpha-gal plates. However, for beta-galactosidase, LacZ gene being not naturally occurring means that the beta-galactosidase is not secreted by yeast, but rather produced in the cell upon activation. Hence, to develop blue phenotype, colony lift assay is necessary as x-gal needs to enter cells (have to permeablise yeast cells first by colony lift assay) for metabolism by beta galactosidase.
For more info on colony lift assay, please refer to www.med.unc.edu > hdohlman > lift_protocol.html

For a clearer demonstration of activation by GAL 4 transcription factor, refer to the diagram below:



Taken from: en.wikipedia.org> wiki > Two-hybrid_screening



To recap, AH109 strain of Saccharomyces cerevisiae contains 4 reporters: ADE, HIS (nutritional markers), LacZ (for beta- galactosidase, artifically cloned from E.coli), MEL1 (for alpha- galactosidase, naturally occuring in some yeast strains).

My previous posts have been focusing on obtaining the bait construct. This is done by cloning in a pcr gene of interest into the bait vector that contains
1) gal4-BD
2) a nutritional factor like TRP marker for yeast selection
3) kanamycin for antibiotic selection
4) Multiple cloning site for cloning purposes.

Once bait construct has been obtained, the plasmid is transformed to yeast strain AH109, and plated on SD/-trp plates for selection of transformed yeast (yeast that is not transformed cannot grow on –trp plates) in 3 serial dilutions of 1/10, 1/100, 1/1000 for calculating transformation efficiency. Once yeast has grown, typically 3-4 days initially when incubated at 30oC, it is restreaked onto fresh -trp plate to form the master plate for mating with the cDNA library, which is commercially obtained. Alternatively, it is possible to clone your own library into a vector, though this is much more of a hassle. cDNA prey library is tittered to find amount of viable cells by plating dilutions of 1/10, 1/100, 1/1000 and 1/10,000 on SD-leu plates (Prey vector contains leu nutritional marker) and making suitable calculations - multiply cfu by plating vol and dilutional factor and divide by total volume available in mL.

Eg, 100ul of 1/10,000 dilution of prey library was plated and gave a cfu of 245 from a total cdna vol of 1ml will yield:
100ul x 10,000 df x 245cfu / 1mL = 2.45x10^8 cfu/mL

Typically, at least 2x10^7 cfu/mL is necessary to produce enough clones for screening in mating (at least 1 million diploids).

If viability of prey is high enough, proceed to mating.

*For easier reference purposes, a table below shows the comparison of bait vector and prey vector:



Mating:

After this, comes the actual mating. Grow out bait colonies in SD/-trp liquid overnight. Next, combine 1mL of bait and prey together in 2x YPDA media (50mL) and incubate at 30oC overnight at 50rpm to ensure cells do not settle down. Check for zygotes after 30hrs under microscope at 40x. If present, pellet at 700g, 5 mins and resuspend into fresh media before plating on 24.5cm x 24.5 cm SD/-trp/-leu/-ade/-his / x-alpha-gal (QDO x-alpha-gal). It is important to note that only mated colonies can grow on QDO (quadruple dropouts as prey provides leu (leucine) marker and bait has trp (tryptophan) marker and interactions between these 2 causes activation of transcriptional factors adenine (ade) and histine (his) as these 2 are under influence of the same promotor.
Within 2-3 days of incubation at 30oC, some colonies may start to turn blue. These colonies are the ones with activation of x-alpha-gal, indicating possible prey protein interaction with bait. These colonies are to be restreaked onto freah QDO x-alpha-gal plates to determine that blue phenotype is not a false positive, which typically turns white after the 2nd streak.

Rescue prey plasmid:

If many colonies are detected, a yeast colony pcr can be used to eliminate same copies of interaction. Upon confirmation, or if there are few colonies for study, yeast plasmid can be extracted by either sonication or using extraction kit (which uses lyticase to break down yeast wall). Plasmid obtain will consist of a mixture of prey, bait and yeast plasmids. To select only prey, the ampicilin selection shall be used – transform to competent cells and plate on amp plates. Bait and yeast plasmids will not be able to grow, hence resultant colonies are all from prey. Miniprep to obtain plasmid from competent cells.

Confirm interaction:

Using the rescued prey plasmid, transform to bait gene and plate to QDO x-alpha-gal plates. If colonies turn blue, use the plasmid obtained from the competent cells for sequencing to determine what protein it is. Blast results using protein query from www.ncbi.nlm.nih.gov > BLAST > blastx

That wraps basically everything in a nutshell. Controls were not discussed as they are too bothersome and the principle is the same. Feel free to ask any questions, though I suggest you guys focus on the mp reports/logbook/whatever else instead and leave me to do the same =)

Cheers,
Debra, TG02

Saturday, 20 October 2007

CHEMISTRY - Measuring Cholesterol

Dear Medical Technologists, I'm going to share with you one of the point-of-care testing system that I've learnt when I was sent out for an Outreach Program few weeks ago. This is regarding cholesterol measurement using the Accutrend® GCT Meter (Roche).

Name of Test: Cholesterol (CHOL) Measurement using Accutrend® GCT Meter




(Picture taken from Divant.com)

Introduction:

Cholesterol is a major steroid in human body. Majority is free, unesterified, that form the structural component of the cell membrane. Its content in the body is dependent on the diet, synthesis within tissue cells and excretion by the liver. Proportion absorbed in the intestine is affected by the amount ingested, with less absorbance when intake is high.

Cholesterol is insoluble in water, thus before being absorbed, unesterified cholesterol is first solublized. It is excreted unchanged or as bile acids in bile. Transportation in plasma and its metabolism are related to lipoprotien metabolism. 2/3 of the plasma cholesterol is in the esterified form.


Materials

  • Accutrend® GCT Meter


  • Accu-Chek Safe-T-Pro Plus® lancet device


  • (Picture taken from: Accu-Chek.ca)

  • Cholesterol Test Strip


  • Code Strip (found in every new pack of test strips, for calibration purpose)


  • Control CH1 (for QC purpose)


  • Alcohol Swab


  • Cotton ball

Procedure (from finger-prick to measurement):


  • Switch on the Accutrend® GCT Meter and insert the test strip


  • Tally the participant's name with the LIS


  • Select the depth of the Accu-Chek Safe-T-Pro Plus® lancet device and the puncture site


  • Clean the puncture site with alcohol swab


  • Twist off the sterility cap and gently press the lancet against the side of the finger tip


  • Apply a hanging drop of blood to the yellow test field on top of the test strip


  • Close the meter flap immediately and wait for 180s countdown


  • Stop the bleed using a cotton ball


  • Read measurement


  • Enter into LIS

Principle of Analysis: Reflectance Photometry

The yellow region of each test strip contains detection reagent which reacts with the blood and causes a colour change. The meter measures the intensity of the reaction colour based on reflectance photometry and calculates the concentration of the sample through a "lot-specific" algorithm (i.e. the data previously entered by mean of code strip), giving the cholesterol reading.

Test Results:

Range: 3.88 - 7.76mmol/L

LO indicates value is below 3.88; HI indicates value is above 7.76

In either cases, turn the strip over and check if the test region is uniformly covered by the blood. If partially coloured, repeat test.

Desirable: < 5.12 mmol/L
Borderline High: 5.12 - 6.18 mmol/L
High: > 6.18 mmol/L

Advantages:

  • Can be performed on non-fasting individual's blood


  • Uses dry chemistry methodology outside a lab setting (suitable for Public screening)


  • Low cost


  • Convenient; Portability


  • Fast results (within 180s)


  • Minimal blood for testing (requires only 1 drop)


  • Easy to operate for the medical worker


  • Gives a relatively accurate result
Disadvantages:

Test is affacted by...


  • IV infusion of Vitamin C


  • Elevated bilirubin (>171umol/L); Jaundice


  • Haematocrit > 55%


  • Methylaminoantiprine


  • Gentisic Acid
Clinical Significance:

There is a good correlation between cholesterol level and incidence of coronary artery diseases. Hence, by offering this fast and convenient point-of-care testing, any risk of atherosclerosis can be detected early. However, further investigation is required to justify the finding.

Questions for me???


PEI SHAN, TG02

Wednesday, 10 October 2007

Haematology

Osmotic Fragility Test

It is used in the diagnosis of hereditary spherocytosis and the screening for thalassemia.
Hereditary spherocytosis is a disorder in which red blood cells are defective because of their round, ball-like (spherical) shape. These cells are more fragile than normal.

Principle:
Red cells will be haemolysed when placed in a hypotonic saline solution. Time and extend of complete haemolysis will depend on the red cell membrane resistance and size and shape of the red cells. Spherocytes will lysed quite quickly in slightly hypotonic solution. Hypochromic microcytic cells will be more resistant to hypotonic solution.

Procedure:
1) Deliver 5ml of 0.9, 0.75, 0.70, 0.65, 0.6, 0.55, 0.50, 0.45, 0.4, 0.35, 0.30, 0.25, 0.2, 0.1% of hypotonic saline solutions into test tubes.
2) 50μl of blood to each tube and mix.
3) Stand tubes for 30mins at room temperature and centurifuge.
4) Pipette the supernatants to cuvette and read absorbance at 540nm.
Note: Supernatent from 0.90% NaCl is used as a blank.

Interpretation of results:
- In normal subjects an almost symmetrical sigmoid curve results.
- Subject with decrease resistance to lysis will show fragility curve that shift to the left.
- Subject with increase lysis will show fragility curve shift to right


(taken from http://www.suite101.com/article.cfm/medical_student/40859)

Clinical Significance
The ability of normal red cell to withstand hypotonicity results from its biconcave shape which allows the cell to increase its volume by about 70% before the surface membrane is stretched, and once this limit is reached lysis occurs.

Spherocytes have an increased volume to surface area ratio; their ability to take in water before stretching the surface membrane is thus more limited than normal and therefore is more susceptible to osmotic lysis.

Decreased osmotic fragility indicates the presence of flattened red cells in which the volume to surface area ratio is decreased. Such a change occurs in iron deficiency anemia and thalassemia in which the red cells with low MCH and MCV are resistant to osmotic lysis.
Eunice
0503245C

Sunday, 30 September 2007

Specimen Reception and Immunology

Hi all,

Sorry for the late posting.
I have just finished Specimen Reception department in my lab and now currently at Immunology.

At the specimen reception department, it is basically about SOPs and following strictly to SOPs. As we have major clients apart from normal clinics, we have to know our clients well because the specimens received from each client are processed differently. So basically, I spent a long time just learning the procedures because we are expected to work like the other staff there.

It is also essential to know the different types of tubes and their functions because we have to inform the doctors and reject the specimen if wrong specimen types are sent for testing. (It has happened quite frequently)

Long Plain tubes:

Known as serum separator tubes when gel is added to the tube. It contains a clot activator (I don't know the exact ingredient) pre-coated in the tube. When spun down the red cells are at the bottom and the gel in the middle with the serum at the top. It is important to note that different type of gels from different materials may cause inaccuracies. For those without the gel, it only has the clot activator. Long plain tubes are usually used for serum analysis such as VDRL testing.

Heparin tubes:

Usually available as sodium, litium or ammonium salt pre-coated in the tube, it is used for trace elements screening in industrial toxicology for patients working in heavily polluting industries. It is also recommended that for potassium analysis, heparin tubes are to be used. When using the long plain, potassium would be released from the platets giving an increased in serum potassium. EDTA tubes comes with potassium salts and would cause falsely high potassium results.

Fluoride/oxalate tubes:

Oxalate will cause osmotic redistribution between the the plasma and erythrocytes, diluting the plasma as a result (water moves from erythrocytes to the plasma ). This would decrease hematocrit levels, disrupt the morphology and cell membranes of erythrocytes. When the samples arrive in our lab, "water condensation" may be observed inside the tube because of the osmotic redistribution. Therefore, fluoride/oxalate tubes are only used for ABO and antibody screening in our lab. Oxalate will also bind to calcium in the blood, causing a falsely low blood calcium level.

Citrate tubes:
Usually for coagulation studies because it preserves the coagulation factors. Citrate also causes osomotic redistribution and decreases the concentration of many analytes (like fluoride/oxalate tubes). However in our lab, citrate tubes are most commonly sent along with a EDTA for full blood count or as a follow up of an EDTA specimen usually due to possible/observed platelet clumping in certain patients (cause unknown) when the specimen is sent in EDTA.


Immunology

During my term in Immunology, the common tests run in the department are:

1. Dengue (most common but low sample volume compared to HIV and it is the only manual test.)

Run by the Centaur machine

2. HIV (most common)
3. Tumor markers ( Ca 199, Ca 125 etc)
4. Ferritin
5. Vitamin B 12 and folate acid
6. Hormones such as cortisol and testosterone

Principles of Dengue is the same as Sally's post.
Principles of HIV is the same as Lizzie's post a number of weeks ago.

As promised, I managed to find out about the alternative system to HIV testing with a much shorter testing time. As it is newly tried out in the Serology department, I don't know much about it yet. It is the Roche Modulla from Roche diagnostics.


Ca 15-3

Ca 15-3 is usually for testing of metastatic breast cancer. When a women is diagnosed with breast cancer, usually, her specimen has the presence of cancer-related antigens especially Ca 15-3. Note that for all tumor marker tests conducted, an abnormal result does not mean that ther patient has cancer. There are also conditions where abnormalities are observed in normal healthy patients. Therefore, a confirmatory test is necessary to detect cancer in patients.
Ca 15-3 can be used to monitor response to treatment of disease. In patient with known metastasis, a reduction may mean a good sign of positive response to treatment while an increase may mean the disease progressing to the next stage.

Principle of the test:
2 step sandwhich ELIZA using direct chemilluminescent.

The system will perform:

1. Dispense sample into cuvette

2. Dispense conjugate reagent and solid phase and incubate at 37 deg celcius.
The conjugate is made of monoclonal mouse antibody 115D8 labelled with fluorescein, which is specific for CA 15-3 antigens. The solid phase is made of monoclonal mouse capture antibody coupled to paramagnetic (refer to PIPC 2 notes for definition of paramagnetic) particles.

3. After incubation, the complex (conjugate and solid phase) is washed to remove unbound antibodies.

4. The complex is resuspended using the wash solution and Lite reagent is dispensed. The Lite reagent consists of monoclonal mouse antibody, DF3, specific for CA 15-3 labeled with acridinium ester.

5. Cuvette is washed to remove unbound Lite reagent.

6. Acid and base reagent is dispensed to initiate the chemiluminescent reaction.

High amount of CA 15-3 in patient's sample = high amount of relative light units (RLU) detected. This means that with an increase in CA 15-3, the intensity of chemiluminescence increases.



Once again, I appologise for posting so late.

With regards,
Yeng Ting
TG02

Cheng Hong: Histology Q&A

Qn1) How many samples are processed for H&E staining? Since it has to be done within 20mins, if something goes wrong surely they do afew samples as a contingency plan rather than banking of a lone sample right?

Ans: normally the pathologist will view at least 2 slides if the margin is not seen clearly due to the cutting, a re-cut must be done.


Qn2)example of a situation in which the doctor is waiting for the results from the lab before stitching up the patient. Sounds like 20 min is a bit rush.

Ans: all of the specimens sent for frozen sectioning are done in the specific time strain and if im not wrong, all of the patients that are waiting for the results are not stitched up yet.


Qn3: What will u do if the slide turn out to have background, pale in the nuclear and cytoplasm?

Ans: A check on the stain is done if needed the stains are changed and a QC slide will be run first before staining the re-cut sample. But normally it wont happen as every morning before the patient’s sections are stained, a QC slide will be run first and check by the senior med-tech.


Qn4:You say that it is importantt to embed the tissue in the correct orientation so i want to ask u what if the orientation is wrong? what are the actions that will be taken by you guys.....another qn is u mention that place slides into the oven set at around 82oC for 15mins(to let the tissue to “stick to the slide” to prevent it from floating off when staining)....well i remember during htech lesson everytime during staining my tissues will get washed off during staining..isit due to insufficient drying...??haf you guys ever encounter tissues being washed off during staining n if yes...wat did u guys do??

Ans:if the orientation that the pathologist request is embedded wrongly it might prevent the pathologist for analyzing the part that is doubtful. So the tissue block must be melted and re-embedded and sectioned again. yes the floating off is due to the insufficient heating and drying. There are times that the tissue might float off so the staining program must be changed eg: let the slides cool down for a minute before staining

Saturday, 22 September 2007

Histology

Cheng Hong: Histology Laboratory

Receiving specimens

1) The specimens will be sent down from the operating theaters/ clinics to the lab by a porter.
2) Upon receiving the specimens the Medical Technologist(MT) must check the total number of bags sent to the lab and check the patient’s particulars and the type of specimen with the request form.
3) Initial on the receiving form and stamp the time.
4) Sign the specimen log book state the number of bags received and the time.
5) Sort out the specimens according to: Placenta/ Point Of Conception(POC)/ Specimens pending for trimming/ Others.
6) The Laboratory Assistant(LA) will then give the specimens a lab assertion number.


Tissue Processing

Specimens for trimming
1) For placenta specimen/ specimens that needs trimming the Pathologist Assistance(PA) will do the trimming and will determine the number of tissue cassettes to use.
2) The PA will dictate the measurements and the characteristics of the specimens to the LA to record
3) The cassettes are then placed in the tissue processing rack and immerse it in buffered formalin.

Product Of Conception
1) POC are stored in sealed packets(to prevent spills/leakages to protect the MT’s safety)
2) POC is processed by the MT: the POC bag is cut into half and the MT must identify the chronic villi and place into cassette.
3) The MT must record the amount of sample used, number of cassette used and if there is any reserve. (Also must indicate if there is any fetal part seen)
4) The cassettes are placed in the tissue processing rack and immerse it in buffered formalin.

Tissue Processor
1) The cassettes in the holder is then placed into the Automated Tissue Processor(Shandon Excelcior).
2) We are able to program the tissue processor to start at a specific time and we are able to choose the different programs(eg:overnight/ rapid etc)
3) The steps are processed as shown below:

1) 10% formalin 30mins(To fix the tissue & preserve cells & tissue constituents)
2) 10% formalin 30mins
3) 70% alcohol 30mins (To remove fixative and water from tissue& replace them with alcohol)
4) 95% alcohol 30mins
5) Absolute alcohol 1hr
6) Absolute alcohol 2hrs
7) Absolute alcohol 2hrs
8) Xylene 30mins (Clearing: Replacing dehydrating fluid with fluid that is miscible with dehydrating fluid & embedding medium)
9) Xylene 30mins
10) Xylene 30mins
11) Paraffin 2.5hrs (wax replacing clearing agent with embedding medium)
12) Paraffin wax 3hrs

*We do not put the tissue into 100% alcohol directly as it will distort the tissue(must be a graded process)

Tissue Embedding
1) After the tissues have been processed, remove the holders and check with the log sheet if the assertion number tallies.
2) Open the cassette carefully and depending on the tissue size, choose a suitable mould size that will fit the tissue so that the entire surface will be exposed when obtaining the sections.
3) Dispense some wax into the mould and place the tissue into the mould and place on the cold plate and press the tissue evenly using a forceps or a pressing block( to ensure all surface will be exposed when sectioning).
4) Place the bottom portion of the cassette on top of the mould and dispense more wax until it fills almost half of the cassette.
5) Allow the wax to cool down for awhile before placing on the ice block(to speed up the hardening of the wax)
*It is important to embed the tissue in the correct orientation so that when the pathologist view the sections it will be in the correct orientation.

*It is also important to be quick when embedding, if the wax hardens before the the tissue is pressed out evenly, problems will arise when obtaining sections.

*For small biopsy, it is important to use a very hot mould so that we can expose the tissue as much as possible to be able to obtain good sections.


Shaving
1) After embedding, the blocks must be shaved first before cutting
2) The MT must be able to know how much to shave and to expose the tissue to prevent shaving off too much of the tissue thus not enough tissue is enough to obtain sections
3) For blocks that have blood clots we can soak them in water for about 5mins to soften the clot for easier cutting
4) For blocks that are fibrous(eg:cervix) we can soak them in softener(commercial softener/ soflant)
5) For blocks that are calcified we can do a surface decalcification by placing it in RDO
6) After steps 2 to 5 we place the blocks on the cold plate before cutting


Paraffin Sectioning
1) The aim is to obtain 3micron thick sections without folds on the tissue.
2) The blocks must be cold to obtain thin sections.
3) Insert the blade and check the thickness setting.
4) Place the block into the tissue block holder and adjust the distance from the blade using the coarse adjustment.
5) Turn the rotary knob away from you to obtain sections.
6) Slowly obtain ribbons and place on water and do any necessary adjustments(removing overlaps/folds).
7) Pick desired section and prepare a clean glass slide and fish up the section.
8) Place the section into a warm water bath(the section will spread out) and fish it up.
9) Let the slide stand for awhile before placing it in the slide holder.
10) Place the rack containing the slides into the oven set at around 82oC for 15mins(this is to let the tissue to “stick to the slide” to prevent it from floating off when staining).

*For sections that need to be cut in levels to slides must be obtain


Routine Haematoxylin and Eosin stain

1) Place the whole slide holder into the Lecia Autostainer and select the respective program
2) The steps as follows below:

(a) Dewax section in xylene for 5 minutes.
(b) Place in another xylene for another 5 minutes.
(c) Rehydrate section with absolute alcohol for 2 minutes.
(d) Place in another absolute alcohol for another 2 minutes.
(e) After then, place in 95% alcohol and 70% alcohol for 1 minute each.
(f) Rinse section with water.
(g) Stain the section with Harris Haematoxylin for 5 minutes.
(h) Rinse section with water.
(i) Differentiate staining with acid alcohol for 5-20 seconds.
(j) Blue the haematoxylin stain with running water or alkaline water.
(k) Check the differentiation microscopically.
(l) Stain the section with eosin for 1 min.
(m) Dehydrate section in graded alcohols with 70% alcohol (10 dips), 90% (10 dips) and 2 absolute alcohols for 3 min each.
(n) Clear the section in xylene for 5 min each with 2 changes.

3) After staining place the slide holder into the auto coverslipper.

*The haematoxylin will stain the nuclei blue or black and eosin will stain the cytoplasm red or pink.

*Eosin is the most suitable dye to combine with alum haematoxylin. It has the ability to distinguish between the cytoplasm of different types of cells and can also distinguish different types of connective tissue fibers.

*Stain results:
Nuclei: blue
Cytoplasm: varying shades of pink
Muscle fibers: deep pinky red
Red Blood cells: orange red
Collagen: pale pinky red
Fibrin: deep red


Frozen Sectioning

1) The lab will receive a call from the Operating Theater(OT) stating the OT number and type of specimen
2) A MT will be sent up to the OT to collect the sample immediately and the pathologist will be informed
3) Upon receiving the specimen, ensure that the patient ID tallies and record the time received and immediately sent back to the lab.
4) Prepare the equipments and tools needed for the pathologist and inform them when it is ready
5) The pathologist will dictate the characteristics and measurements of the specimen and mark out the orientation of the specimens using different colour dyes.
6) About 1 to 2 samples will be cut from the specimen into size about 15mm length and with and about 3to4mm thick.
7) Squeeze some freezing medium on to the tissue holder and place the tissue on it and immerse it into liquid nitrogen for about 5to8 seconds.
8) Place the tissue holder with the frozen tissue into the freezing microtome and shave off some tissue to obtain a full face.
9) Cut the tissue and using a glass slide to pick up the section( the temperature difference of the tissue(-35oC) and the glass slide(RT) will cause the freezing medium to melt and adhere to the slide.
10) Proceed to Rapid Staining


Rapid Staining
1) Xylene 10dips
2) Wash in running water
3) Haematoxylin 1min
4) Wash in running water until no more haematoxylin runs off
5) Ammonia 3 to 5 dips
6) Wash in running water
7) Eosin 10dips
8) 70% alcohol 10dips
9) 95% alcohol 10dips
10) 100% alcohol 10dips
11) Xylene 10dips
12) Xylene 10dips
13) Mount with DEPEX
14) Slide viewed by pathologist and the pathologist will call the doctor in the OT about the diagnosis.

*The whole process must be done within 20mins as the patient in the OT is still unstitched, the doctor must know if they have removed all the tumor from the body.