Medical Microbiology- dPBL- Case 1 to 6- Microorganisms Identifications

Case 1 (Suat Fang, 0503328G)

Particulars of patient

Name: Khong Fay Seah Sex: Female
HRN: OPD 009 IC No. : S00055X
Date of birth : 1/12/80 Age : 27 years
Ward/Clinic : Clinic M Bed No : -------

Clinical diagnosis

Complains: Fever, chills and dysuria
Diagnosis: Urinary Tract Infection
Specimen: Urine

Suspected Microorganisms Identifications



References

http://en.wikipedia.org>wiki>Enterococcus
http://en.wikipedia.org>wiki>Escherichia_coli
http://en.wikipedia.org>wiki>Klebsiella_pneumoniae
http://en.wikipedia.org>wiki>Proteus_mirabilis
http://en.wikipedia.org>wiki>Pseudomonas_aeruginosa
http://en.wikipedia.org>wiki>Staphylococcus_saprophyticus
http://www.austincc.edu>microbugz>html http://www.bact.wisc.edu>themicrobialworld>E.coli.html
http://www.bd.com>ds>technicalCenter>inserts>L007359(08)(1206)
http://www.umdnj.edu>micrsweb>case2gramnegatives>intro.html

Case 2 (Sharifah, 0503189C)
Particulars of patient

Name : Kwan Siew Yan Sex : Female
HRN : OPD001 IC No. : S000123X
Date of birth : 16/6/78 Age : 23 years
Ward/Clinic : Clinic X Bed No : -------

Clinical diagnosis

Complaints: Diarrhea
Diagnosis: Enterocolitis
Specimen: Stool

Suspected Microorganisms Identifications

References
http://en.wikipedia.org>wiki> Salmonella
http://en.wikipedia.org>wiki> Escherichia_coli
http://en.wikipedia.org>wiki> Campylobacter
http://en.wikipedia.org>wiki> Vibrio
http://en.wikipedia.org>wiki> Yersinia
Levinson, W. (2004). Review of Medical Microbiology and Immunology. 9th edition. McGraw-Hill.

Case 3 (Royston, 0503289A)
Particulars of patient

Name : Maisy Hong Sex : Female
HRN : 006789T IC No. : S000111Y
Date of birth : 1/12/40 Age : 67 years
Ward/Clinic : AB2 Bed No : -------
(Patient is in-patient, thus more susceptible to nosocomial infection)

Clinical diagnosis

Complaints: Fever, chills, bladder distension (stretching); on indwelling catheter
Diagnosis: Urinary Tract Infection
Specimen: Urine

Suspected Microorganisms Identifications

References

http://www.wikipedia.org>agar_plates
http://www.wikipedia.org>Enterococcus
http://www.wikipedia.org>Escherichia_coli
http://www.wikipedia.org>Klebsiella
http://www.wikipedia.org>Proteus
http://www.wikipedia.org> Pseudomonas_aeruginosa
http://www.wikipedia.org>Staphylococcus_saprophyticus

Case 4 (Najib, 0503217B), (Charmaine, 0503186I)

Particulars of patient

Name : Tong Wei Hong Sex : Male
HRN : OPD 004 IC No. : S000444X
Date of birth : 1/12/39 Age : 68 years
Ward/Clinic : Clinic M Bed No : -------

Clinical diagnosis

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

Suspected Microorganisms Identifications

References

http://www.blinn.edu>natscience>phillips>Micro%20Pictures.htm
http://www.hpa-standardmethods.org.uk>documents>bsopid>pdf>bsopid11.pdf http://www.mc.maricopa.edu~johnson>labtools>Dbiochem>opto.html
http://www.pubmecentral.nih.gov>articlerender.fcgi?artid=379757
http://www.sigmaaldrich.com>img>assets>13860>75744.pdf

Case 5 (Jeremy, 0503168G)
Particulars of patient

Name : Wong Fei Hong Sex : Male
HRN : OPD 0010 IC No. : S210444X
Date of birth : 1/12/70 Age : 37 years
Ward/Clinic : Clinic S Bed No : -------

Clinical diagnosis

Complaints: Fever, swelling around operation wound
Diagnosis: Wound infection
Specimen: Wound swab

Suspected Microorganisms Identifications

References
Warren Levinson. Review of medical microbiology and immunology (9^thed).
http://users.stlcc.edu/kkiser/biochem.html
http://www.oxoid.com

Case 6 (Natalie, 0503275J)
Particulars of patient

Name : Ong Fei Fei Sex : Female
HRN : OPD 0013 IC No. : S210334X
Date of birth: 1/12/70 Age: 37 years
Ward/Clinic: Clinic T Bed No: -------

Clinical diagnosis

Complaints: Fever, pain during urination, virginal discharge
Diagnosis: UTI
Specimen: Virginal Discharge

Suspected Microorganisms Identifications

Escherichia coli (E.coli)

Test: Gram-Stain
Biochemical Tests: Lactose fermentation test, Indole test, Lysine test
Treatment: Antibiotics which may be used to treat E. coli infection include (but are not limited to) amoxicillin as well as other semi-synthetic penicillins, many cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin and the aminoglycosides

Chamydia

Test: Gram-Stain/DNA-based test
Identification: Chlamydia species are readily identified and distinguished from other chlamydial species using DNA-based tests. Most strains of C. trachomatis are recognized by monoclonal antibodies (mAbs) to epitopes in the VS4 region of MOMP
Treatment: It may be treated with any of several antibiotics such as azithromycin, erythromycin or doxycycline/tetracycline.

Klebsiella pneumoniae

Test: Gram Stain
Biochemical Tests: Indole test, citrate test, urease test, motility test, malonate test, Phenylalanine slant test
Treatment: carbenicillin, ampicillin, quinolones, and ceftazidime

Pseudomonas aeruginosa

Test: Gram Stain
Biochemical Tests: Triple Ion Sugar (TSI), Oxidase test, Indole test, Citrate test
Treatment: Aminoglycosides, Quinolones, Cephalosporins, Ureidopenicillins

Followings are the additional microorganisms of other causative agents of UTI:

Candida albicans (yeast)
Candida albicans is a diploid fungus (a form of yeast), which is capable of mating but not of meiosis, and a causal agent of opportunistic oral and genital infections in humans. C. albicans is among the gut flora, the many organisms which live in the human mouth and gastrointestinal tract. Under normal circumstances, C. albicans lives in 80% of the human population with no harmful effects, although overgrowth results in candidiasis.

Test: Calcofluor- white Gram Stain followed by direct microscopy, Culturing
Treatment: Amphotericin B, Ketoconazole

Trichomonas vaginalis (protozoan)
Trichomonas vaginalis, an anaerobic, parasitic flagellated protozoan, is the causative agent of trichomoniasis, and is the most common pathogenic protozoan infection of humans in industrialized countries.

Test: Pap smear, Culturing
Treatment: Metronidazole/ Tinidazole

Gardnerella vaginalis
Gardnerella is a genus of gram-variable bacteria of which Gardnerella vaginalis is the only species. Gardnerella vaginalis can cause bacterial vaginosis in some women.

G. vaginalis is an aerobic, non-motile, slow growing coccobacillus. It grows as small, circular, convex, gray colonies on chocolate agar; it will also grow on HBT agar. A selective medium for G. vaginalis is colistin-oxolinic acid blood agar

Test: Microscopy of Clue cells, Amine test
Treatment: metronidazole

Illustrations for the types of Biochemical tests used and their results

1. Citrate Utilisation Test: The citrate tube is used to determine if an organism is capable of utilizing citrate.

2. Urease test: The urea agar slant allows detection of urease activity of both rapidly urease pos organisms as well as enterobacteriaceae family.

3. Motility Test: Used to detect the motility of organisms in a semi-solid gelatin medium.

4. Indole Test: Can be detected by its ability to combine with certain aldehyde to form a coloured compound.

5. Phenylalanine Slant Test: Determine the ability of an organism to deaminate phenylalanine to phenylpyruvic acid enzymatically with resulting acidity.

6. Malonate Broth Test: Determine the ability of an organism to use sodium molante as the sole carbon with resulting alkanity and it is used to differentiate enterobact family.

Triple Sugar Iron (TSI) Test: Used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates.

All pictures extracted from: http://users.stlcc.edu/ >kkiser>biochem

Laboratory Investigations (Bacteria Identification)

The specimen (virginal discharge) will be cultured so as to isolate the microorganisms short listed above. Next, a series of biochemical tests will be carried out and incubated for a day before verifying the results for the final identification of the specific microorganism. Lastly, antibiotic susceptibility testing is performed to achieve a cure for the infection.

1) Gram-staining followed by microscopy examination (of unknown bacteria)
i.e. Gram Negative: E. Coli, P. Aeruginosa, and Enterobacter-Klebsiella-Serrtia Family

2) Culturing of bacteria
MacConkey’s Agar: To check for Lactose Fermenters
Positive: E.Coli and Enterobacter-Klebsiella-Serrtia Family
Negative: P. Aeruginosa
Nutrient Agar: To observe for P. Aeruginosa
Eosin- methylene blue Agar: E. Coli

3) For gram-neg bacteria, biochemical tests are carried out (i.e. different tests + F12). F12 is of antimicrobial agents such as penicillin, it tells us the sensitiveness and susceptibility of the bacteria towards different types of antibodies.

Example of Biochemical Tests:

1. Simmons citrate


2. Urease


3. Motility (& OF) test


4. Indole


5. Phenylalanine slant


6. Malonate test

References

http://en.wikipedia.org>wiki>Escherichia_coli
http://en.wikipedia.org>wiki>Chlamydia_trachomatis
http://en.wikipedia.org>wiki>Pseudomonas_aeruginosa
http://en.wikipedia.org>wiki>Klebsiella_pneumoniae
http://en.wikipedia.org>wiki>Trichomonas_vaginalis
http://en.wikipedia.org>wiki>Gardnerella
http://en.wikipedia.org>wiki>Candida_albicans
http://kidney.niddk.nih.gov>Kudiseases>pubs>utiadult/
http://whitewolf.newcastle.edu.au>techinfo>proc_bacto_biochem
http://www.healthscout.com>Gardnerella Vaginalis>Symptoms

Medical Microbiology- dPBL- Case 1 to 6

Case 1 (Suat Fang, 0503328G)
Particulars of patient

Name : Khong Fay Seah Sex : Female
HRN : OPD 009 IC No. : S00055X
Date of birth : 1/12/80 Age : 27 years
Ward/Clinic : Clinic M Bed No : -------

Clinical diagnosis

Complains: Fever, chills and dysuria
Diagnosis: Urinary Tract Infection
Specimen: Urine

Introduction

The lower urinary tract, which contains the bladder and urethra, and the upper urinary tract, that contains two kidneys and the ureters, makes up the 2 sections of urinary tract.

UTI is the infection of one or more components of the urinary tract due to bacteria that enter the opening of the urethra. Urine does not normally contain microorganisms. When bacteria get into the bladder or kidney and multiply in the urine, they cause a UTI. UTI is more common in women because their urethra is shorter and closer to the anus.

Suspected microorganisms

o Escherichia coli (E.coli),
o Klebsiella pneumoniae,
o Proteus mirabilis
o Pseudomonas aeruginosa
o Staphylococcus saprophyticus and
o Enterococcus spp.

Escherichia coli (E.coli)
· gram-negative bacterium
· found in the digestive tract
· Present on the skin around the rectal area
· typically ferment lactose
· grows well on MacConkey agar
· Most common cause of UTI.

Stain: gram staining
Biochemical Test: Oxidase test, MRVP

Klebsiella pneumonia
· gram-negative
· non-motile
· lactose fermenting
· facultative anaerobic
· rod shaped bacterium
· found in the normal flora of the mouth, skin, and intestines
· Second common cause of UTI.

Stain: gram staining
Biochemical Test: Oxidase test, Indole-Test

Proteus mirabilis
· gram-negative
· facultative anaerobic bacterium
· shows swarming, motility, and urease activity
· rod shaped bacterium
· Has the ability to produce high levels of urease. Urease hydrolyzes urea to ammonia (NH3) and thus makes the urine more alkaline.

Stain: gram staining
Biochemical Test: Oxidase test, indole test

Pseudomonas aeruginosa
· gram-negative
· motile
· aerobic rod shape bacteria
· oxidase-positive
· do not ferment lactose
· common inhabitants of soil and water
· tend to cause disease in humans with abnormal host defenses.

Stain: gram staining
Biochemical Test: Triple Ion Sugar (TSI)

Staphylococcus saprophyticus
· gram-postive
· facultative anaerobes
· coagulase-negative species of Staphylococcus bacteria
· catalase-positive
· reside in the urinary tract and bladder of sexually active females.
· phosphatase-negative
· urease and lipase positive.

Stain: gram staining
Biochemical Test: Catalase test, coagulase test

Enterococcus faecalis
· gram-positive Streptococci
· spherical bacterium which forms pairs or chains during growth

Stain: gram staining
Biochemical Test: Catalase test, PYRase activity test

1. The urine sample will be cultured on blood agar and Cystine-Lactose-Electrolyte Deficient (CLED) agar and incubated.
2. Gram-staining will then be done to differentiate between gram-positive and gram-negative microorganisms.
3. If the gram-stain showed gram-positive cocci, the catalase test can be done to differentiate between Streptococcus and staphylococcus bacterium.
4. Coagulase test will be performed if catalase test shows positive results to determine the type of Staphylococcus bacterium.
5. PYRase activity test can be done for negative results catalase test to determine the type of Streptococcus bacterium.

References

http://en.wikipedia.org>wiki>Enterococcus
http://en.wikipedia.org>wiki>Escherichia_coli
http://en.wikipedia.org>wiki>Klebsiella_pneumoniae
http://en.wikipedia.org>wiki>Proteus_mirabilis
http://en.wikipedia.org>wiki>Pseudomonas_aeruginosa
http://en.wikipedia.org>wiki>Staphylococcus_saprophyticus
http://kidney.niddk.nih.gov>Kudiseases>pubs>utiadult>
http://www.healthassist.net>conditions>uti.shtml
http://www.medicinenet.com>urine_infection>article.htm

Case 2 (Sharifah, 0503189C)
Particulars of patient

Name : Kwan Siew Yan Sex : Female
HRN : OPD001 IC No. : S000123X
Date of birth : 16/6/78 Age : 23 years
Ward/Clinic : Clinic X Bed No : -------

Clinical diagnosis

Complaints: Diarrhea
Diagnosis: Enterocolitis
Specimen: Stool

Introduction

- Definition
‘Enterocolitis’ is the combination of two words ‘Enteritis’ which is the inflammation of the small intestine and ‘Colitis’ which is the inflammation of the large intestine, specifically, the colon.

- Characteristics
It is characterized by the inflammation of the epithelial and subepithelial tissue at the small and large intestines. Symptoms include presence of blood in feces, abdominal pain and diarrhea (as complained by this patient).

Suspected Micro-organisms

- Salmonella
- Shigella
- Campylobacter
- Escherichia
- Vibro
- Yersinia

Salmonella
- Gram negative rod-shaped bacterium
- Motile
- Produces Hydrogen Sulphide
- Non- lactose fermenter
- Has a high infectious dose (must have a high dose to cause infection)
- Is an invasive organism
- Typical species that causes enterocolitis (however, other species are known to have causes said disease as well) : Salmonella typhimurium

Shigella
- Gram negative rod-shaped bacterium
- Non-motile
- Do not produce Hydrogen Sulphide
- Non- lactose fermenter
- Has a low infectiouse dose
- Is an invasive organism
- Usually involved in bacillary dysentery
- Typical species that causes enterocolitis : Shigella dysenteriae, Shigella sonnei

Campylobacter
- Gram negative rod, comma or S-shaped bacterium
- Motile
- Microaerophilic (grows best in 5% Oxygen)
- Usually involved in bacillary dysentery
- Typical species that causes enterocolitis : Campylobacter jejuni

Escherichia
- Gram negative rod, comma-shaped bacterium
- Motile
- Do not produce Hydrogen Sulphide
- Lactose fermenter
- Is an invasive organism
- Typical species that causes diarrhea : Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC)

Vibrio
- Gram negative rod, comma-shaped bacterium
- Motile
- Is a slow lactose fermenter
- Found in marine organisms
- Halophiles
- Is not an invasive organism
- Typical species that causes diarrhea : Vibrio cholerae, Vibrio parahaemolyticus

Yersinia
- Gram negative rod-shaped bacterium
- Motile
- Non- lactose fermenter
- Is not an invasive organism
- Transmitted by fecal contaminations by domestic animals
- Typical species that causes enterocolitis : Yersinia enterocolitica

Types of Preliminary Tests to be Performed

- Microscopy (Gram Stain)
- Leukocyte Count
- Stool Occult Blood
- Culture
o MacConkey
o Blood
o XLD or DCA
o TCBS
- Enrichment Broth
o Selenite

Types of Secondary Tests to be Performed

- TSI Slant
- Oxidase Test
- Indole Test
- Fermentation of Sugars (e.g. Glucose, Lactose, Mannitol etc.)

References

Levinson, W. (2004). Review of Medical Microbiology and Immunology. 9th edition. McGraw-Hill.
http://en.wikipedia.org>wiki>Salmonella
http://en.wikipedia.org>wiki>Campylobacter
http://en.wikipedia.org>wiki>Escherichia_coli
http://en.wikipedia.org>wiki>Vibrio
http://en.wikipedia.org>wiki>Yersinia

Case 3 (Royston, 0503289A)
Particulars of patient

Name : Maisy Hong Sex : Female
HRN : 006789T IC No. : S000111Y
Date of birth : 1/12/40 Age : 67 years
Ward/Clinic : AB2 Bed No : -------
(Patient is in-patient, thus more susceptible to nosocomial infection)

Clinical diagnosis

Complaints: Fever, chills, bladder distension (stretching); on indwelling catheter
Diagnosis: Urinary Tract Infection
Specimen: Urine

Introduction

Given the symptoms from the case, the UTI is most like to be upper urinary tract infections like pyelonephritis, which is an ascending urinary tract infection that has reached the pelvis of the kidney.

Background Info: Catheter-associated UTI
Causes
A catheter is a hollow tube that is used to drain urine from the bladder. An indwelling catheter stays in place for long periods of time. The presence of a catheter within the urinary tract increases the likelihood of urinary tract infection. As the urinary catheter is left in place for long periods of time, bacteria will inevitably grow in it. A harmful infection may occur if the number of bacteria becomes large or if specific pathologic bacteria grow in the urinary tract.

Signs and Tests
1. A dipstick test to detect the presence of nitrites and substances produced by bacteria that caused UTIs. Hence, a positive test indicates that an infection is present before urine cultures are performed.
2. A urinalysis may show white blood cells (WBCs) or red blood cells (RBCs).
3. A urine culture maybe performed to determine the type of bacteria in the urine and the appropriate antibiotic for treatment.

Collection of urine specimen: As an indwelling catheter is in place, the urine should be obtained by sterile aspiration of the catheter with needle and syringe but not from the collection bag.

Guidelines for indwelling catheter urine specimen
1. Do not collect urine from the drainage bag because growth of bacteria outside the catheter may have occurred at this site.
2. Clean the catheter with an alcohol pad.
3. Use a sterile needle and syringe to puncture the tubing. Aspirate the urine directly from the tubing.
4. Transfer the urine to a sterile specimen container.
5. Urine catheter tip cultures are not acceptable.

Suspected Micro-organisms
· Enterobacteriaceae species
o E. coli
o Klebsiella-Enterobacter-Serratia
o Proteus-Providencia-Morganella
· Staphylococcus saprophyticus
· Streptococci species (Entercocci)
· Pseudomonas aeruginosa

Key characteristics of micro-organisms
1) Enterobacteriaceae species
• Gram negative rods
• Facultative anaerobes
• Catalase positive
• Oxidase negative

1a) E. Coli
• Rapidly ferment lactose
• Beta-hemolytic
• Produce positive indole test
• Positive for b-glucoronidase using the substrate
• Ferments mannitol

1b) Klebsiella-Enterobacter-Serratia

Klebsiella species
• Non-motility
• Lysine carbohydrate positive
• Citrate positive
• Have large polysaccharide
• Voges-Proskauer positive
• Rapidly ferment lactose

Enterobacter species
• Motile
• Citrate positive
• Ornithine decarboxylase positive
• Voges-Proskauer positive
• Rapidly ferment lactose

Serratia
• Produces Dnase, lipase and gelatinase
• Voges-Proskauer positive
• Slow fermenter of lactose

1c) Proteus-Providencia-Morganella
• Does not ferment lactose
• Motile
• Grow on potassium cyanide medium
• Ferment xylose
• Urease positive for Proteus species and Morganella morganii
• Urease negative for Providencia species

2) Staphylococcus saprophyticus
• Gram positive cocci arranged in grape-like clusters
• Catalase positive
• Coagulase negative
• Phosphatase negative
• Urease & lipase positive

3) Streptococci species (Entercocci)
• Gram positive cocci arranged in pairs
• Facultative anaerobes
• Catalase negative
• Non-hemolytic
• Bile-esculin positive
• Able to grow in 6.5% NaCl

4) Pseudomonas aeruginosa
• Gram negative motile rods as single/pairs/occasionally short chains
• Oxidase positive
• Does not ferment lactose

References

Geo FB, Janet SB & Stephen AM. (2004). Jawetz, Melnick, & Adelberg’s Medical Microbiology. 23rd edition. McGraw-Hill.
http://en.wikipedia.org > search
http://www3.umdnj.edu/ >micrsweb>case2gramnegatives>intro.html

Case 4 (Najib, 0503217B), (Charmaine, 0503186I)
Particulars of patient

Name : Tong Wei Hong Sex : Male
HRN : OPD 004 IC No. : S000444X
Date of birth : 1/12/39 Age : 68 years
Ward/Clinic : Clinic M Bed No : -------

Clinical diagnosis

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

Introduction

Bronchitis is a respiratory disease where the mucous membrane of the lungs’ bronchial passage is inflamed. The swollen membrane narrows and shut off the tiny airways in the lungs, causing cough that is accompanied with thick phlegm and breathlessness.

The disease comes in two forms: acute (lasting less than 6 weeks) and chronic (reoccurring frequently for more than two years) bronchitis. Acute bronchitis is commonly caused by lung infections where 90% of the infections are of viral origin and the remaining 10% of bacterial origin. Chronic bronchitis may be caused by one or more factors and this include repeated attacks of acute bronchitis which will weaken and irritate bronchial airways over time.

Suspected microorganisms

Influenza A and B
Parainfluenza virus
Moraxella catarrhalis
Haemophilus influenzae
Chlamydia pneumoniae
Pseudomonas aeruginosa
Streptococcus pneumoniae

4 bacteria were short listed for this case:

Streptococcus pneumoniae (S. pneumoniae):
S. pneumoniae are Gram-positive, lancet-shaped cocci (elongated cocci with a slightly pointed outer curvature). They are usually seen as diplococci, but they may also occur singly and in short chains. Individual cells are between 0.5 and 1.25 micrometers in diameter. They do not form spores, and they are non-motile. They lack catalase and ferment glucose to lactic acid

Stain: Gram stain
Biochemical Test: MR-VP test

Pseudomonas aeruginosa (P. aeruginosa):
P.aeruginosa is a Gram-negative, aerobic and rod-shaped bacterium with no particular arrangement. Although it is classified as an aerobic organism, P.aeruginosa is considered by many as a facultative anaerobe as it is well adapted to proliferate in conditions of partial or total oxygen depletion. Adaptation to anaerobic environments is essential for certain lifestyles of P. aeruginosa, like during lung infection in cystic fibrosis patients where thick layers of alginate surrounding bacterial mucoid cells can limit the diffusion of oxygen.

Stain: Gram stain
Biochemical Test: Triple sugar iron (TSI)

Moraxella catarrhalis (M. catarrhalis):
M. catarrhalis is a gram negative, aerobic, oxidase-positive diplococcus which may colonise and cause respiratory tract associated infections in humans.

Stain: Gram stain

Haemophilus influenzae (H. influenzae):
H. influenzae is a non-motile Gram-negative coccobacillus. It is generally aerobic, but can grow as a facultative anaerobe. The organism is also catalase and oxidase positive.

Stain: Gram stain

Investigation

The sputum will be cultured so as to isolate the microorganisms short listed above. Then the biochemical tests specific to each bacteria will be performed so as to identify them.

References

http://en.wikipedia.org>wiki>Haemophilus_influenzae
http://en.wikipedia.org>wiki>Moraxella_catarrhalis
http://en.wikipedia.org>wiki>Pseudomonas_aeruginosa
http://en.wikipedia.org>wiki>Streptococcus_pneumoniae
http://www.medicinenet.com>bronchitis>page3.htm
http://www.nlm.nih.gov>medlineplus>bronchitis.html

Case 5 (Jeremy, 0503168G)
Particulars of patient

Name : Wong Fei Hong Sex : Male
HRN : OPD 0010 IC No. : S210444X
Date of birth : 1/12/70 Age : 37 years
Ward/Clinic : Clinic S Bed No : -------

Clinical diagnosis

Complaints: Fever, swelling around operation wound
Diagnosis: Wound infection
Specimen: Wound swab

Introduction

For a wound to be considered as a surgical site infection, it must fufill the following criteria:
- Infection must occur within 30 days of surgery
- Infection must involve only the skin and subcutaneous tissue
- Must be at least one of the following:
o Purulent discharge from a superficial infection OR
o Organisms isolated from aseptically wound culture
- Must have at least one of these signs:
o Pain or tenderness
o Localised swelling
o Redness or heat

Surgical wound infection is caused by endogenous or exogenous.
An example of endogenous infection is due to poor surgical technique while an example of exogenous infection is due to improper sterilization of instruments.

Suspected microorganisms
Staphylococcus aureus (facultative anaerobe, gram-positive cocci)
Enterococcus faecalis (facultative anaerobe, gram-positive cocci)
Streptococcus pyogenes (facultative anaerobe, gram-positive cocci)
Escherichia coli (facultative anaerobe, gram-negative bacilli)
Pseudomonas aeruginosa (aerobic, gram-negative bacilli)
Clostridium species (anaerobic, gram-positive bacilli)
Enterobacter species (facultative anaerobe, gram-negative bacilli)
Proteus mirabilis (facultative anaerobe, gram-negative bacilli)
Klebsiella pneumoniae (facultative anaerobe, gram-negative bacilli)

5 bacteria were short listed for this case:

Staphylococcus aureus (S.aureus):
S. aureus is a Gram-positive, cluster-forming cocci. Human are the major reservoirs of S.aureus. They are non-motile, non-spore forming facultative anaerobes. They are able to ferment mannitol, and are catalase and coagulase positive. S.aureus is the common microorganism present in surgical-wound infection.

Stain: Gram stain
Biochemical Test: Catalase test, coagulase test, mannitol, DNase test

Enterococcus faecalis (E.faecalis):
E.faecalis is a Gram-positive, facultative anaerobic cocci. Along with E.coli, they are indicators for faecal contamination. E.faecalis have also emerged as a significant, antibiotic-resistant, nosocomial pathogen.

Stain: Gram stain
Biochemical Test: MRVP

Streptococcus pyogenes (S.pyogenes):
S.pyogenes is a Gram-positive, facultative anaerobe cocci. They are non-motile, non-sporeforming cocci that occur in chains or in pairs of cells. They are normal flora of the body but can cause infection after penetrating the host defence.

Stain: Gram stain
Biochemical Test: Catalase test, coagulase test, mannitol

Escherichia coli (E.coli):
E.coli is a Gram-negative, facultative anaerobe bacillus. E.coli can grow in the presence or absence of O2. Under anaerobic conditions it will grow by means of fermentation, producing characteristic "mixed acids and gas" as end products. However, it can also grow by means of anaerobic respiration, since it is able to utilize NO3, NO2 or fumarate as final electron acceptors for respiratory electron transport processes. E.coli is a normal flora of the body, and can be found in intestines and feces of human.

Stain: Gram stain
Biochemical Test: MRVP

Pseudomonas aeruginosa (P.aeruginosa)
P.aeruginosa is a motile, Gram-negative, aerobic bacillus. P.aeruginosa is an opportunistic pathogen in human. They exploit any break of defense in human to cause an infection. It is primary a nosocomial pathogen.

Stain: Gram stain
Biochemical Test: Oxidase test

Investigation

The swab will be cultured so as to isolate the microorganisms short listed above. Then the biochemical tests specific to each bacteria will be performed so as to identify them.

References

http://www.surgical-tutor.org.uk>default-home.htm?principles>microbiology>wound_infection.htm
http://www.textbookofbacteriology.net>e.coli.html
http://www.textbookofbacteriology.net>normalflora.html
http://www.textbookofbacteriology.net>pseudomonas.html
http://www.textbookofbacteriology.net>staph.html
http://www.textbookofbacteriology.net>streptococcus.html

Case 6 (Natalie, 0503275J)
Particulars of patient

Name : Ong Fei Fei Sex : Female
HRN : OPD 0013 IC No. : S210334X
Date of birth : 1/12/70 Age : 37 years
Ward/Clinic : Clinic T Bed No : -------

Clinical diagnosis

Complaints: Fever, pain during urination, virginal discharge
Diagnosis: UTI
Specimen: Virginal Discharge

Introduction

Urinary Tract Infection (UTI) is commonly suspected in clinical practice and up to 50% of all women may suffer from symptomatic UTI at some time during their lives. UTI is considered to be complicated when it affects pregnant women, children, men or the elderly and if it affects kidney tissue (Upper UTI). While simple UTI is uncommon in men aged 20-50, prostatic enlargement in older men may cause urinary tract obstruction and thereby causing UTI.

Normally, urine is sterile. An infection occurs when tiny organisms, usually bacteria from the digestive tract, cling to the opening of the urethra and begin to multiply. The urethra is the tube that carries urine from the bladder to outside the body. Most infections arise from one type of bacteria, Escherichia coli (E. coli), which normally lives in the colon.

In many cases, bacteria first travel to the urethra. When bacteria multiply, an infection can occur. An infection limited to the urethra is called urethritis. If bacteria move to the bladder and multiply, a bladder infection, called cystitis, results. If the infection is not treated promptly, bacteria may then travel further up the ureters to multiply and infect the kidneys. A kidney infection is called pyelonephritis.

Suspected microorganisms

Escherichia coli (E.coli)
Chlamydia
Mycoplasma
Klebsiella pneumoniae
Proteus mirabilis
Psedonmonas aeruginosa,

4 bacteria were short listed for this case:

Escherichia coli (E.coli)
Escherichia coli (E. coli), is a Gram-negative, non-sporulating, facultative eubacterium that is commonly found in the lower gastrointestinal tract of warm-blooded animals. Peritrichous strains are motile, but some strains lack flagella. E.coli are not always confined to the intestine, and their ability to survive for brief periods outside the body make them an ideal indicator organism to test environmental samples for fecal contamination.

As Gram-negative organisms, E. coli are resistant to many antibiotics that are effective against Gram-positive organisms.

Test: Gram-Stain
Treatment: Antibiotics which may be used to treat E. coli infection include (but are not limited to) amoxicillin as well as other semi-synthetic penicillins, many cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin and the aminoglycosides

Chlamydia
Chlamydia trachomatis is one of three bacterial species in the genus Chlamydia, family Chlamydiaceae, class Chlamydiae, phylum Chlamydiae, domain Bacteria. C. trachomatis is a gram-negative bacteria. It comprises two human biovars: trachoma and lymphogranuloma venereum (LGV). Many, but not all, C. trachomatis strains have an extrachromosomal plasmid.

Test: Gram-Stain/DNA-based test
Identification: Chlamydia species are readily identified and distinguished from other chlamydial species using DNA-based tests.Most strains of C. trachomatis are recognized by monoclonal antibodies (mAbs) to epitopes in the VS4 region of MOMP
Treatment: It may be treated with any of several antibiotics such as azithromycin,erythromycin or doxycycline/tetracycline.


Klebsiella pneumoniae
Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines. it is distinguished by being indole-negative and by its ability to grow on both melezitose and 3-hydroxybutyrate. It naturally occurs in the soil and about 30% of strains can fix nitrogen in anaerobic condition.

Test: Gram Stain
Biochemical Test: Indole-Test, Melezitose Test

Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium. Almost all strains are motile by means of a single polar flagellum. P. aeruginosa secretes a variety of pigments, including pyocyanin (blue-green), fluorescein (yellow-green and fluorescent, now also known as pyoverdin), and pyorubin (red-brown). P. aeruginosa is often preliminarily identified by its pearlescent appearance and grape-like odor in vitro. Definitive clinical identification of P. aeruginosa often includes identifying the production of both pyocyanin and fluorescein as well as its ability to grow at 42°C. Although classified as an aerobic organism, P. aeruginosa is considered by many as a facultative anaerobe as it is well adapted to proliferate in conditions of partial or total oxygen depletion. This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, and in its absence it is also able to ferment arginine by substrate-level phosphorylation.

Test: Gram Stain
Biochemical Test: Triple Ion Sugar (TSI)

Investigation

The specimen (virginal discharge) will be cultured so as to isolate the microorganisms short listed above. Next, a series of biochemical tests will be carried out and incubated for a day before verifying the results for the final identification of the specific microorganism.

References

http://en.wikipedia.org>wiki>Chlamydia_trachomatis
http://en.wikipedia.org>wiki>Escherichia_coli
http://en.wikipedia.org>wiki>Klebsiella_pneumoniae
http://en.wikipedia.org>wiki>Pseudomonas_aeruginosa
http://kidney.niddk.nih.gov>Kudiseases>pubs>utiadult>
http://www.medicinenet.com>urine_infection>article.htm

Results of my MTT assay.

Hey guys ive been talking alot on MCT stuff and MTT assay. Now its time for me to show the fruits of my labour...........the RESULTS!!!

Cell Sensitivity (MTT) assay

Cool ah?? Hahha. With this result, i will find the IC50 of the drug against the Hep G2 cells.

IC50 or the half maximal inhibitory concentration, represents the concentration of the inhibitor (drug) that is required for 50% inhibition of its target (Hep G2 cells). IC50 measures how much the drug is required for 50% inhibition in-vitro.

I will take a minimum of 5 points to form a linear line. Then using Microsoft Excel, i will obtain the equation of line and calculate the IC50.








With the equation, substitute the y value with 50 and then the x value could be obtained and that is the IC 50 of drug towards Hep G2 cells.

Enlightened?? Hahhaha See you guys in sch.


Najib Bin Hamid
(0503217B)

Preparation of a Hanging Drop

this is a simple experiment to observe the movement of microorganisms under microscope.

Materials:
- bacteria sample
-cover slip
-glass slides
-plasticine
-inoculating loop
-microscope

Method:

1. Use plasticine provided; make a loop about the size of a 5 cent coin.
2. Place the loop firmly onto the center of a clear glass slide.
3. Aseptically transfer a loop of sample broth culture onto the center of the clean cover slip.
4. Place the glass slide (with the plasticine loop facing downwards) over the cover slip and turn the glass slide over. The drop of culture must appear to hang down from cover slip.
5. View 10X and 40X

Movement of E. Coli was observed. i thought the E. Coli will be swimming, but actually movement of E. Coli under microscope is just vibrating in the original position.

Difficulty faced during experiment is when i was expected to turn the glass slide over, the E. Coli tends to slide to the side of the plasticine. it was only after a few tried that i finally managed to do it right.

that's all!

Suat Fang
0503328G

Plant subcultuing

Hi guys! I'll be blogging on something that we do not usually have the chance to do in our curriculum! During this period, I'm like a gardener, the only difference is that I'm working in a plant lab and not a garden.. :)

Okie! Now about plant subculturing...

A subculture is actually a culture made by transferring tissue from a previous cultures to a fresh medium in order to prolong the life of a particular strain where there is a tendency to degeneration in older cultures. A plant subculture in many ways are similar to a cell subculture in terms of aseptic techniques involved, characteristic of a subculture and precautions to be taken.

Before a subculture can be carried out, the media used should be autoclaved at 121 deg cel for 15 min in order to kill off any microorganisms. After the media is autoclaved, the container should not be open unneccessary as it is prone to contamination. If necessary, it can only be opened in a horizontal flowhood which confers more product protection.

The selected plant culture has to be in a healthy state and free of contamination. The whole subculturing process have to be done in the horizontal flow hood to minimize contamination. Forceps and scissors should all be sterilized before use. The flow hood should also be UV and swapped with 70 % ethanol before use. Strict aseptic techniques applies. The containers is opened by applying force onto the four edges of the containers with thumbs and index fingers only. Once the plant container is opened, hands with gloves should not be placed over the plant culture. Only sterilized scissors and forceps are allowed to touch any of the plant culture, media or within the containers. The lower portion of the stem are cut and retrieved out using a forcep. All the leaves are then removed, using 2 forceps. Hence by removing the leaves, the nod of the stem is exposed in which new plantlets will grow from them. The stem is then inserted lying horizontally into the media with the use of forceps. the stems are aranged in organized manner (in rows) so as to prevent overcrowding.

Finally the plant subculture is ready for growth in the growth room. Nevertheless, the plant subculture are supplied with 12 hours of light daily and should be constantly check for contamination. Once sufficient plant subculture is grown, it is havested for use.

feel free to ask any question!

Royston Tan
0503289A

Food Sampling

haha... i am so sorry guys... looks like we are all mixed up with the dates... i shall blog now too... poor nat got so much questions to answer...

alright. back to the basics. as u all know, i am still attached to the food and water lab. so now, i shall take all of u on an excursion on how i collect my samples! (food sampling)

For our lab, we do sample collection from hotel's restaurants. To go and collect samples, we first need to prepare some items.

Ice Box with an Ice Pack - this is to lower the temperature of the food to slow down or minimise the multiplication of the microorganisms. Swab the ice box with beacoup first!

Sterile spoons - To collect samples

Sterile bag - To store samples

Marker - To label the time, date, place of collection, the name of the sample and person collecting.

Empty bag - To dispose the used spoons.


After packing all the necessary stuff, we get to sit in the company's car to the hotel!

When we reach the hotel, we proceed to find the chef in charge. They will provide us with the samples that we need to collect for testing.

When collecting samples, always use the sterile spoons. DO NOT use tongs or utensils provided by the hotel as they might have been contaminated. ALWAYS practice aseptic techniques when collecting food. DO NOT touch the food sample onto any outer surface of the sterile bag as it is already exposed to the environment. After collection, seal the sterile bag up. Write the time, nature, date of sample. Write also the restaurant's name and the name of the person who collected it. After that, store the food in the ice box. Seperate the raw food and cooked food with the ice pack.

After collecting all the samples, we are now ready to head back to the laboratory. While waiting for the driver, we get to take a look around the hotel!

Back at the laboratory! place all the food samples into the refrigirator and store until the time when the food is going to be tested!

Thats all! hope you enjoy this post! lol... see you guys in school soon!

~Jeremy~
TG01

Hi all! I'll be touching on an assay that should be familiar to all of us ---- Bradford Assay.

Bradford Protein Assay is actually a simple and accurate procedure that is used to determine the concentration of protein in solutions.

Bradford assay is a protein determination method that involves the binding of Coomassie Brillant Blue dye to proteins. The dye exists in 3 forms : cationic (red), neutral (green) and anionic (blue). The dye is usually in the protonated red cationic form. However, when the dye binds to protein, it is concverted to a stabe unprotonated blue form. It is this blue dye form that is detected at 595 nm in the assay using an ELISA plate spectrophotometer.

Usually a standard bovine serum albumin (BSA) (2mg/ml) is used to plot the standard curve that will be used to extrapolate the protein concentration of the samples. Various concentrations of the standards BSA will be prepared as follows:

Concurrently, the protein samples are also diluted, usually in a 20x dilution. Once these are prepared, the tubes are then vortex to allow the well mixing and centrifuged to bring down all the proteins.

For my experiments, both the standards and samples are pipetted into a 96-well plate in triplicates, which means that for each standard or sample, there will be 3 wells. 250ul of Coomassie Brillant Blue dye aka Bradford reagent is then added in. Take into consideration that this step is time critical because bradford reagents are light sensitive. Therefore, very often, an aluminium foil will be used to cover the 96-well plate after the addition of the reagents.

When the absorbance are given by the spectrophotometer, a standard graph of absorbance value against protein concentration is plotted. An example of the graph is shown :

With this standard curve, the protein concentration can be extrapolated.

One thing to note when plotting the graph is the R-squared value. The desired R-squared value is 1. However, it is not possible to get this value unless there are only 2 standards. Therefore, for my lab, as long as the R-squared value is greater than 0.99, the graph is accepted and the protein concentration that is extrapolated from this graph is said to be reliable and accurate. If the desired R-squared value of greater than 0.99 cannot be achieved, the assay will be carried out again.


Thats all for bradford assay! Its just sweet and simple! Hope u guys understand! Feel free to ask me any qns!

Take care and see ya all in 2 weeks time!

Charmaine
TG01

Reticulocyte Count

Hi all.
I am kinda lost with the shedule on whoose gona blog this week or what, so i'll just post something okay (: sorry to the one who ought to blog this week! HA.

Okay, i shall introduce Recticulocyte Count Test that is done in the Haematology Section;

1) Recticulocyte Count (RC) (in another words, rectic count)

Introduction: The recticulocyte count is used in the evaluation of anemia as it accurately reflects the amount of erythrocytes production taking place in the bone marrow.
Relationship btwn anaemic condition and erythrocyte production in bone marrow (in normal cases):
anaemic condition= increase in RBC production therefore increase of RC in blood
However, if the RC is not raised, it shows an indictation of impaired bone marrow function or lack of eythrocytes stimulus.

Principle: Recticulocytes are junvenile red cells. Thus they contain remnants of the ribosome and the ribonucleic acid, which are present in larger amt in the cytoplasm of the nucleated precursors from they are derived.
New Methylene Blue or Brilliant Cresyl Blue are supravital dyes that are used to measure reticulocytes.
Currently, there are 2 methods being employed:
a)Manual Method: The use of Brilliant Cresyl Blue
b) Automated method: Reticulocytes Package by Cell Dyn Ruby analyzer
We used the automated method cause its more efficient and contributes to a faster Turn Around Time (TAT).

Procedure of Automated Method

1) When using the retoculocyte reagent, verify the expiration date and store the stock reagent in the dark at room temperature
2) Label patient accession No. on to the tube of reticulocyte reagent
3) Verify that the whole blood specimen is warmed to room temperature and well mixed pior to
sampling
4) Pipette 20 uL of the blood sample into each labelled tube of reticulocyte reagent
5) Incubate the stained Reticulocyte specimens on a rotator or in a rack, after fully inverting the stained specimens 4-6 times. Inbubation must be performed according to the reagent package insert.

Note: The stained Reticulocyte specimens must incubate for at least 15mins but no more than 2 hours pior to processing on the Cell Ruby machine.

Although the process is time-efficient, there are some limitations of the procedure.

Okay im done with elaborating. Hm i shall list some definations of defined abnormalities (that we 'kinda' need to know and understand in the haema section)


Leukocytosis is an elevation of the white blood cell count (the leukocyte count) above the normal range. The normal adult human leukocyte count in peripheral blood is 4.4-10.8 x 109/L. A white blood count of 11.0 x 109/L or more suggests leukocytosis.
Leukocytosis is very common in acutely ill patients. It occurs in response to a wide variety of conditions, including viral, bacterial, fungal, or parasitic infection, cancer, hemorrhage, and exposure to certain medications or chemicals including steroids. Leukocytosis can also be the first indication of neoplastic growth of leukocytes.
For lung diseases like pneumonia,tuberculosis etc. WBC count are very inportant for the diagnosis of the disease that means leucocytosis can be seen in above mentioned diseases

Neutrophilia is a condition where a person has a high number of neutrophil granulocytes in their blood.
Neutrophils are the primary white blood cells that respond to a bacterial infection, so the most common cause of marked neutrophilia is a bacterial infection.
Neutrophils are also increased in any acute inflammation, so will be raised after a heart attack or other infarct.
As well as increasing in number, neutrophils show other changes in infection and inflammation.
A neutrophilia might also be the result of a malignancy. Chronic myelogenous leukemia(CML or chronic myeloid leukaemia) is a disease where the blood cells proliferate out of control. These cells may be neutrophils. Neutrophilia can also be caused by appendicitis.

Lymphopenia is the condition in which there exists an abnormally low number of lymphocytes in the blood.
Lymphopenia can be caused by various types of chemotherapy, such as with cytotoxic agents or immunosuppresive drugs. Some malignancies in the bone marrow will also cause lymphopenia.
A decreased number of lymphocytes (notably T cells) is present in those with AIDS. People exposed to large doses of radiation, such as those involved with Chernobyl can also exhibit a lymphopenia.
Lymphopenia may be present as part of a pancytopenia, when the total number of blood cells are reduced. This can occur in marrow failure.

Monocytosis is an increase in the number of circulating monocytes. In humans, 950/μL is regarded as at the upper limit of normal; monocyte counts above this level are regarded as monocytosis.
Monocytosis often occurs during chronic inflammation. Diseases that produce this state:
Infections: tuberculosis, brucellosis, listeriosis, subacute bacterial endocarditis, syphilis, infectious mononucleosis and other viral infections and many protozoal and rickettsial infections (e.g. kala azar, malaria, Rocky Mountain spotted fever).

Eosinophilia is the state of having a high concentration of eosinophils (eosinophil granulocytes) in the blood. The normal concentration is between 0 and 0.5 x 109 eosinophils per litre of blood. Eosinophilia can be reactive (roughly, allergic) or non reactive.
Diseases that feature eosinophilia: Hypereosinophilic syndrome, Parasitic infections, allergic disorders.
The release of interleukin 5 by T cells, mast cells and macrophages stimulates the production of eosinophils
Anemia is a deficiency of red blood cells (RBCs) and/or hemoglobin. This results in a reduced ability of blood to transfer oxygen to the tissues, causing tissue hypoxia.
The three main classes of anemia include excessive blood loss (acutely such as a hemorrhage or chronically through low-volume loss), excessive blood cell destruction (hemolysis) or deficient red blood cell production (ineffective hematopoiesis).

Basophilia is an uncommon cause of leukocytosis. Basophils are inflammatory mediators of substances such as histamine. These cells, along with similar tissue-based cells (mast cells), have receptors for IgE and participate in the degranulation of white blood cells that occurs during allergic reactions, including anaphylaxis
Causes; Infections: viral infections (varicella), chronic sinusitis Inflammatory conditions: inflammatory bowel disease, chronic airway inflammation, chronic dermatitis
Thrombocytosis is the presence of high platelet counts in the blood, and can be either reactive or primary (also termed essential and caused by a myeloproliferative disease).
High platelet counts can occur in patients with polycythemia vera (high red blood cell counts), and is an additional risk factor for complications.

p.s. the definations above are just F.Y.I okaaay.

See yall soooon! (:

Natalie
TG01