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

Dengue NS1 Antigen Strip

Hi Everyone! Goodness, I can't believe our SIP is ending soon. I'm already getting so used to working in a clinical lab. Heh.

Anyways, today I will be blogging about another testkit. I know I keep blogging about testkits la but at my workplace, almost everything is automated, even the pre-analytical stuff. Thus, I get very little hands-on tests to perform. Other than testkits, of course =).

Correct me if i'm wrong but I think somebody has already blogged abt the Dengue Duo Cassette which is a testkit for Dengue Serology (IgG/IgM). Soo, I will blog about the Dengue Antigen NS1. Yay.

Name of Test: Dengue Antigen Detection/ Dengue NS1 Ag Detection

Introduction:
Singapore has recently been hit with a dengue plaque with the number of cases rising steadily to about 13 000 and 19 already succumbed to this illness. Common indications include a high fever and a hematology report stating a sudden, steep drop in platelet count with or without an increased hematocrit. The patient may also have dengue antibody (IgM for first infection or Igm and IgG for secondary and subsequent infection) present in the serum. During the acute phase of dengue, when the antibody titer is too low to be detected using testkits, the NS1 antigen is used as a marker. This antigen is present from the onset of the fever and remains in the serum up to 9 days after the onset of fever.

Principle, Method and Result Interpretation:

• The method adopted in this testkit is lateral flow immunochromatography. The testkit comes with 25 test strips and a migration buffer.
• The test strip itself has membrane that was formed with a sample pad, a conjugate pad and a membrane where the result lines will be displayed.
• The sample pad is for loading the sample. The conjugate pad contains gold colloidal particles that are coated with anti-NS1 monoclonal antibodies and also gold colloidal particles coated with streptavidin.
  
• Method:
• 50 microliters of the sample and 1 drop of migration buffer is dispensed into a small glass tube.
  
• A strip is placed into the glass tube in an upright position in the correct orientation and incubated for 15 minutes.
  
• Should the Control Line not appear after 15minutes or appear faint and doubtful, re-incubate for a further 15minutes and interpret the results.
  
• If the Control line fails to appear after 30 mins of incubation in total, discard the strip and repeat the test with a new strip.
• Results:
• If present, the NS1 antigen will bind with the gold colloidal particles coated with anti-NS1 antibodies as the sample migrates up the strip (aided by the migration buffer) to the conjugate pad. As it reaches the Result membrane, a blue or purple line will appear.
• The Control line should appear as biotin from the migration buffer reacts with streptavidin to form a blue or purple line at the Result membrane.
• A positive result is indicated with 2 lines on the result membrane.
• A negative result is indicated with 1 line on the result membrane as the Control Line.
  
• This test can be used in conjuction with other laboratory data to aid in diagnosis of dengue even though it has a few limitations.
  

Clinical Significance:
In the laboratory, this test is used as a rapid test to detect the presence of NS1 antigen in serum. Previously, this was a test sent out to our referral laboratory where RT-PCR was used to determine the presence of NS1 Antigen. This test is a useful qualitative test that significantly helps in the diagnosis of dengue during this dengue plague in Singapore because it is a test that produces an accurate result quickly. This, in turn, aids in ensuring proper treatment for the patient more efficiently.

Cheers,
Sharifah
TG01

Technical Stuff

Hello Everybody! Sorry for disappearing for a long long time! Today, I'm back! I'm going to blog about plant subculturing which I did for my SIP. It's quite boring though.

In plant subculturing, small pieces of plant tissues are placed on or in a media rich in nutrients and sugar. The major media components are made up of some or all of the followings :

- Macronutrients
- Micronutrients
- Vitamins
- Amino acids or other nitrogen supplements
- Sugar(s)
- Other undefined organic elements
- Solidifying agents
- Growth regulators

Macronutrients :
Macronutrients provide the 6 major elements : nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) required for plant cell or tissue growth. Culture media should contain at least 25-60 mM inorganic nitrogen for adequate plant cell growth. Potassium is required in nitrite, sulfate or chloride form at concentration of 20-30 mM. Optimal concentration of phosphorus, magnesium, sulfur and calcium range from 1-3 mM.

Micronutrients :
Essential micronutrients for plant cell growth include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu) and molybdenum (Mo). Chelated form of iron and zinc are commonly used at minute concentration of uM. Iron maybe the most critical of all the micronutrients.

Vitamins :
Normal plants synthesize the vitamins required for their growth and development. Vitamins are required by plants as catalyze in various metabolic processes. As the plants are cultured in vitro, vitamins may become limiting factors for cell growth. The vitamins most frequently used are thiamine (B1), nicotinic acid, pyridoxine (B6) and myo-inositol. Myo-inositol although it is a carbohydrate not a vitamin, has been shown to stimulate growth. Therefore it is commonly included in many vitamin stock solution at concentration of 50-5000 mg/L.

Amino acids or other nitrogen supplements :
Although culture cells are capable of synthesizing all of the required amino acids, the addition of amino acids may be used to further stimulate cell growth. Amino acids provide plant cells with an immediately available source of nitrogen which generally can be taken up by cells more rapidly than inorganic nitrogen. The most common source of inorganic nitrogen are casein hydrolysate, L-glutamine, L-asparagine and adenine.

Sugar(s) :
Carbohydrates must be supplemented to the culture medium as few cell lines are fully autotropic i.e., capable of supplying their own carbohydrate needs by CO2 assimilation during photosynthesis. Carbon and energy source of plant cell culture media is sucrose. Glucose and fructose maybe substitute in some cases, glucose being as effective as sucrose and fructose being somewhat less effective. The concentration of sucrose normally ranges between 2-3%.

Other undefined organic elements :
Addition of a wide variety of organic extracts to culture media often results in favourable tissue response. Supplements include protein hydrolysate, coconut water, yeast extracts, malt extracts, ground banana, orange and tomato juice. However, undefined organic supplements should be only be used as a last resort. The addition of activated charcoal (AC) to culture media may have beneficial effect. The effect of AC is generally attributed to one of the three factor : absorption of inhibitory compounds, absorption of growth regulators and darkening of the medium.

Solidifying agents :
Agar is the most commonly used gelling agent for preparing semi-solid and solid plant tissue culture medium. First, when agar is mixed with water, it forms a gel that elts at approximately 60-100 deg C and solidify at 45 deg C; thus, agar gel are stable at all incubation temperatures. Another geling agent commonly used is Gelrite. This is a product of bacterial fermentation and should be used at 1.25 - 2.5 g/L, resulting in a clear gel which aids in the detection of contamination.

Growth regulators :
Four broad classes of growth regulators are important in plant tissue culture; the auxins, cytokinins, gibberellins and abscisic acid. Both auxin and cytokinin are usually added to culture media in order to obtain morphogenesis, although the ratio of hormones required is not universally the same. The type of morphogenesis that occurs in a planbt tissue culture largely depends upon the ratio and concentration of auxin and cytokinin present. Root initiation of plantlets, embryogenesis and callus initiation all generally occur when the ratio of auxin to cytokinin is high, whereas shoot proliferation occurs when the ratio is low.

An example of a general media receipe :

Growth regulator : MS (Murashige and Skoog) 6.45g
Vitamin : Myo-inositol 30mL
Organic elements: No name 30mL
Sugar(s): Sucrose 45g
pH 5.8 -5.81
Activated charcoal 1g

Top up to 3L with distilled water

And thats the end of my boring entry this time! Feel Free to ask any questions and sry if I bore u too much! muahahaha...

Royston Tan
0503289A
TG01

Cell lines and MTT assay

Eloooooooooooo. This week i am going to share with you guys about the cell lines that i am using for my project. For all the cell lines, i had to perform MTT assay aka cell proliferation assay. I will further explain what MTT assay is later in the post.

Firstly lets talk about the cell lines. For my project, I am using 8 human cancer cell lines and they are:

1. A431 (skin cancer)
2. HepG2 (liver cancer)
3. Chang Liver (liver cancer)
4. A427 (lung cancer)
5. CRL-2119 (pancreas cancer)
6. MDA-MB-231 (breast cancer)
7. CCL-244 (colon cancer)
8. CCL-220.1 (colon cancer)

To learn the full profile of the cancer cell lines and also the difference between the same cancer (eg. the 2 types of colon cancer), go to http://www.atcc.org/.

Based on the recommended medium (from atcc.org) ,

A431 is grown in DMEM (Dulbecco's modified Eagle's medium)

HepG2, Chang liver and A427 are grown in MEM (minimal essential media)

CRL-2119 and MDA-MB-231 are grown in a 1:1 mixture of DMEM and Ham's F12 medium

CCL-244 and CCL-220.1 are grown in ATCC-formulated RPMI-1640 Medium

Now I will talk about MTT assay.

MTT assay is a laboratory test and a standard colourmetric assay for measuring cellular proliferation. The assay measures the changes in colour and it is used to determine cytotoxicity of potential medicinal agents such as drugs.

Method to perform MTT assay(assay is done in 4 days)

1. Discard spent media from culture flask into waste bottle
2. Wash with 10mL PBS and discard
3. Add 2ml of trypsin and incubate for 2- 3 minutes
4. After cells are detached, add 8mL of media to deactivate the trypsin and mix well
5. Transfer 10mL of cell suspension into a 50mL centrifuge tube
6. Centrifuge the cell suspension at 1500 rpm for 3 minutes
7. Discard supernatant and resuspend cell pellet while washing with 10mL PBS
8. Centrifuge at 1500 rpm for 3 minutes and discard supernatant
9. Resuspend the cells in 5mL-10mL of fresh medium
10. Transfer 50mL of cell suspension into an eppendorf tube and add 50mL of trypan blue
11. Add 20uL of the stained cells to a hemocytometer and perform a cell count
12. Prepare cells to obtain a final no. of 1 X 10^4 cells / 100uL by dilution if necessary
13. Seed 1 X 10^4 cells into each well of the 96 well plate 9 (groups of 5)
14. Incubate overnight at 370C, 5% CO2 incubator
15. Next day, add 1uL of drug* into each well
16. Incubate overnight at 370C, 5% CO2 incubator
17. Next day, add 25uL of MTT reagent into each well
18. Incubate for 2 hours at 370C, 5% CO2 incubator
19. After 2 hours, add 100uL of cell lysis buffer into each well
20. Incubate overnight at 370C, 5% CO2 incubator
21. Next day, obtain the results from multiplate reader (absorbance at 570nm)

*Drug used is cis-Platinum(II) diammine dichloride of different concentrations

The yellow MTT reagent is reduced to purple formazan in the mitochondria of living cells. Then a solubilization solution (cell lysis buffer) is added to dissolve the insoluble formazan product into a coloured solution. The reduction process of formazan takes place only when mitochondrial reductase enzymes are active and therefore conversion is directly related to the number of viable cells. When the amount of purple formazan produced by cells treated with an agent(drug) is compared to the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of the cells can be deduced through the production of a dose response curve.

Ill end here for now.

Najib Bin Hamid (0503217B)

Testing on Escherichia coli

Hello everyone! I am sorry about the late posting again. Was doing my project until I forgot about the blog posting until today.

Another common test in our laboratory is the detection of Escherichia coli. From Ying Ying’s first posting, we know that is LTB tubes are positive (gas production), we will have to continue by inoculating a loop of the positive LTB tube into a BGB and a EC tube.

If the food sample is positive for EC tube (gas production), we can conclude that the bacterium is a lactose fermenter. Further tests to confirm whether it is Escherichia coli needs to be carried out.

A loopful of the culture from the EC tube is being streaked onto an Eosin Methylene Blue agar.
Eosin Methylene Blue agar (EMB)
- Consists of Eosin Y and Methylene Blue dyes which inhibit gram-positive bacteria growth and differentiates lactose fermenting and non-lactose fermenting bacteria
- Lactose fermenting bacteria will appear blue-black
- Non-lactose fermenting bacteria will appear colourless and transparent.
- If positive for Escherichia coli, purple colour colonies with green metallic sheen will grow.

E.coli on EMB plate



After incubating for 1 day at 35◦C, the EMB plate is taken out. If positive for Escherichia coli, purification of a single colony for the EMB is done and incubated.

After purification, biochemical tests are being carried out.

Indole test
- If positive for Escherichia coli, a red ring is being formed when Kovac’s reagent is added. This is because Escherichia coli produces tryptophanase which cleaves tryptophan to produce indole and other products. Kovac’s reagent will then react with indole to form a red ring. (Positive indole test)

Methyl Red test
- If positive for Escherichia coli, the methyl red indicator will turn red due to the acids produce by the degradation of glucose, lowering the pH. (Positive MR test)

Voges-Proskauer test
- If positive for Escherichia coli, there will be no change. This is because E.coli does not produce acetyl methyl carbinol which will react with alpha-napthol and potassium hydroxide to produce pink colour after standing for about 1 hour. (Negative VP test)

Simmon’s Citrate test
- If positive for Escherichia coli, there will be growth on the surface of the Simmon’s Citrate slant but no colour change from green to blue.
Gram-stain
- If positive for Escherichia coli: gram-negative bacillus. (pink, rod-shaped)






















Gram stain of E.coli




Results of the biochemical tests are then recorded and compared with the results of the control to check whether it is positive for Escherichia coli.

~Jeremy~

TG01

Biological Indicators

Hi all.

Sterilization Monitoring Using Biological Indicators

In order to ensure that the autoclave machine is functioning properly and sterilizing appropriately, biological indicators will be used. Biological indicators are contains a spore disc inoculated with bacterial spores (Geobacillus stearothemophillus) for steam sterilization and a culture medium, containing a pH indicator, encased in a crushable, glass ampoule. The acid production associated with growth causes a change in colour of the biological indicator. This aids in the detection of growth.

Methods

1. Place one or more vials in the most difficult location to sterilize and run the cycle. Usually at 121°C for 15mins.
2. Seal the cap immediately after retrieving the vials by pressing down firmly until flush with tube.
3. Allow the tubes to cool. Purpose of cooling is to prevent bursting of ampoule due to heat and pressure.
4. Crush the media ampoule by squeezing the sides of the tube.
5. Ensure that the spore disc has been completely saturated with the growth media. If not, gently tap it on a hard surface until disc is totally immersed in the media.
6. Incubate tubes at 55°C for 48 hours, checking at 24hrs interval. A control should be incubated together with those sterilized tubes.

Results

Control: Yellow

Sterilized Vials: Purple

If the vials that were sent for sterilization turn yellow (original colour is purple), it shows that the autoclaving/ sterilization process failed.

that's all! =)

Suat Fang
tg01
0503328G

Overdue answers to all your questions..Sorry.

Eloo guys. Sooooooooooo Sorry that I took such a long time to answer all your questions. I totally forgot about them until Ms Chew reminded me. Sorry again. Below are the answers to the questions. Hope it will help. All your postings are very interesting and I learnt a lot form all the experiences that you guys blog about.

To Randall
For my lab, if there is any contamination, I would just check the microbes responsible for the contamination. If it is a bacterial infection, I simply add sodium hypochlorite (NaClO) before disposing them as general waste. Fungal infections are the most dangerous among all, as the spores are very hardy and could spread easily. Therefore for a fungal infection, the CO2 incubator will be disinfected.
In my lab, there are a lot of frozen cell lines. Lab stuff also frequently freezes cells. Therefore if there were any contamination, the cultures will be disposed into the biohazard bin and new cell line will be thawed.
If there are still contamination occurring, I will culture the media without any cells or add PBS to the media to check if the media contains any microbes.

To Charmaine
Yup I wash twice with PBS so that for the second wash, all the trypsin will be removed from the media. Like I said in my post, washing with PBS for the second time will decrease the possibility for the cells to be resistant towards the trypsin as trypsin is totally removed in the second washing step. Yup I pipette the cells in the centrifuge tube.

To Eugene
I have 8 cancer lines and they all need different types of media. Each media contains different components. Usually I will just go to the webpage where my lab bought the cells and refer to the recommended media for the particular cell line. The media used in my lab are pre-made by the supplier. Then before using the media, I have to add 10% FBS and antibiotics to the media. I will elaborate on which cell line gets which media during my next post.

To Yong Young
For my lab, there are no limits to the number of passages as I am using cancer cell lines. Cancer cell lines are more hardy and have the machinery to over come cell death. (immortal cell lines). But there’s always a but, even though I can subculture as many times I want for cancer cell lines, I have to check for its morphology as they might revert back to the normal, uncancerous state through series of mutations. I havent encountered any mutations though. If such things happen, I have to discard the particular cell and thaw new cells.

To Shu Hui
Trypsin is an enzyme that will help to dislodge the cells that are attached on the surface of the culture flask. It helps to re-suspend those adherent cells from the culture flask. With this, we are able to subculture and count the cells for other experiments or assays.

To Lizzie
For me, if there is a colour change in the media, I will check the culture under the inverted microscope. If there are cells floating (dead cells), I will change the media as soon as possible. Sometimes media colour changes are also due to contamination. Usually, I will change the media once the colour in a way that is very different form the normal colour of the media. For example I will change media if the media turns orange yellow or yellow. I will not change if the media is orangy-red and there are no floating cells found.

To Zahirah
There are other dyes that can be used for cell counting. I will give you an example of one that I researched on. It is know as a Fluorescent dye. This method indirectly counts the number cell by counting the nucleus with the help of a machine known as the NucleoCounter (an integrated fluorescence microscope designed to detect signals from the fluorescent dye). The dye, propidium iodide (PI) bound to the DNA of cell nuclei. The NucleoCounter counts the total cell count and viability of cell samples such as mammalian, yeast, somatic, fish and sperm cells.

To Ye Tun
I will elaborate more on the cell lines I use for my next post.
In my lab, I use the CO2 incubator system. To me manually adding buffers (done in your lab) to maintain the pH of the culture risk more contamination as you have to expose the culture when adding the buffers. In my lab I use ventilated caps for my culture flasks. Which means, I do not need to unscrew the cap a little to allow gasses to exchange in the CO2 incubator thus minimize the risk of contamination. The ventilated caps pores are made specially small so that no microbes could enter.

To Suat Fang
I will elaborate and explain on MTT assay on my next post.


Have fun ppl.

Najib Bin Hamid
(0503217B)

Question and Answer.

hello guys.

shocked to see a post from me right?!
hmm. im here because, on last friday i was asked to answer a qn that was asked by ms chew.
and finally, i've found the answer. (:


Heres the qn:
Why is there detection of the hemoglobion and blood (2 different parameters instead of just one since they are all RBCs) in the urine dipstick analysis?

Brief Answer:
Cause of RBCs/Hb in urine:
Erythrocytes in the blood transport oxygen from the lung to the tissues by binding it to haemoglobin. Excretion of unphysiological amounts of erythrocytes into the urine - i.e. more than about 130.000/24 h - is most often caused by inflammation or other lesions in the urogenital tract. Free hemoglobin originating from lysed erythrocytes (intravascularily or in the sample during storage/transport) may be found in the urine as well. This can be due to a variety of other diseases. Hemoglobin passes into the urine when the binding capacity of the plasma and the tubular re-absorption capacity have been exceeded. This usually occurs with plasma hemoglobin concentrations of around 60 µmol/l.

The interpretation of results: The practical detection limit of the test pad for blood of the Combur-Test® urine test strips is about 5 erythrocytes/µl and for hemoglobin the amount corresponding to 10 erythrocytes/µl. A homogeneous color change indicates the presence of lysed erythrocytes or free hemoglobin in the urine specimen (hemoglobinuria).
Green dots will show up if intact RBCs cells are present on the test paper (hematuria).
Reference range: 0 - 5 erythrocytes/µl

Difference between Hemoglobinuria & Hematuria
Hemoglobinuria is a condition in which the oxygen transport protein hemoglobin is found in abnormally high concentrations in the urine. The condition is often associated with hemolytic anemia, in which RBCs are destroyed, thereby increasing levels of free plasma hemoglobin. The excess hemoglobin is filtered by the kidneys, which release it into the urine, giving urine a red colour.
Hematuria is the presence of blood in the urine. It is a sign of a large number of diseases of the kidneys and the urinary tract, ranging from trivial to lethal.
p.s. Occasionally "hemoglobinuria" is used synonymously, although more precisely it refers only to hemoglobin in the urine.

Interpretation
Transient micro-hematuria
-Heavy physical training or exhausting activities
-Urinary tract infections
-Trauma to the kidneys or urinary tract
Permanent or recurrent micro-hematuria
-Urolithiasis
-Tumours of the kidneys or the urinary tract
-Glomerulonephritis
-Pyelonephritis
Medicines possibly causing micro-hematuria
Phenytoin, rifampicine, danazole, anticoagulants including acetylic salicylic acid, NSAIDs like ibuprofen, cytostatics like cyclophosphamide


Hope it clears the doubt of yall too! (: seeya!
Natalie
TG01

Microbiology

Hello children!!!!

Haiz, so refreshing to go back to school for a change. Nonetheless, i must admit that i'm getting a little too used to the ole working life. Haha. I know some of you already are enjoying yourselves riiight.

So anyway, its been 10weeks (no way man!) and I've been posted to almost all the benches in my lab - urinalysis, microbiology, hematology, blood banking, order entry and clinical chemistry. Next month, i might even get a chance to follow the phlebotomists on their rounds to the wards and observe how they draw blood. Super cool. And and! Recently, Pei Shan (we're in the same lab) & I underwent a Point-of-Care Testing training as we will be sent out to do an Outreach Programme next month at a shopping mall. Ultra cool. So I guess that means, I can go back to school and apply my finger-pricking skills to my classmates. Mwahaha.

I'm sorry, I'm too used to digressing. So this week, i'm going to blog about a Stool Toxicological test using a testkit called ImmunoCard.

Name of Test: Detection of Clostridium difficile Toxin A & Toxin B in Stool

Introduction:
Clostridium difficile is a species of gram-positive rod-shaped bacteria that is the main cause in infectious diarrhea. Most strains of this bacteria produce 2 biologically and immunologically different toxins: Toxins A & B. A rapid test such as this ImmunoCard test enables the physician to verify any possible infection quickly and begin treatment immediately.

Principle:

• This is a rapid enzyme immunoassay
• Consists of a membrane held in a plastic frame with 2 sample ports and 2 reaction ports. The left-hand side column represents the Control panel while the other column represents the Test panel.
  

• This membrane contains immobolized antibodies to Toxins A & B. An Enzyme Conjugate, Wash Buffer, Substrate Reagent and Specimen Diluent is also provided along with the kit. The Enzyme conjugate contains antibodies to Toxins A&B coupled with horseradish peroxidase. The diluent is a buffered protein solution.
• Patient stool sample is diluted with the Specimen Diluent and the Enzyme Conjugate is added to it. It is then incubated for 5 mins. During this time, if there is toxin present, the molecules of the toxins will bind to the antibodies in the Enzyme Conjugate.
• Once incubation period is over, an aliquot of the sample is added onto the sample ports and incubated for another 5mins at RT. This time, the toxin-enzyme conjugate is separated from any particulate matter as it seeps through the membrane to the Test and Control reaction Ports.
• The toxin-conjugate complexes are captured in the Test port by immobolized anti-toxin. Then, the Wash Reagent, which is also a buffered similar to the Sample Diluent, is added to both ports. The reaction ports are then incubated again for 5 mins. At this point in time, the enzyme (HRP) modifies the Substrates Reagent, causing a colour change.
• The test kit is then read visually after incubation. The Control Port should be blue in colour while the Test Port with a blue colouration indicates presence of Toxins.

Test Results:
As this is not a quantitative test, there are no reference ranges. Test results will only indicate the presence of absence of Toxins A&B of C. difficile.

A positive result is indicated by a blue colouration in 2 Reaction Ports. A blue colouration in the Control Reaction Port (upper left) means that the sample has been added, reagents were active at the time of use and that proper sample migration has occurred.

If there is only blue colouration on the Control Reaction Port (upper left) and the Test Reaction Port (upper right) is colourless, then this would be a negative result.

Clinical interpretation:
As mentioned this test is to determine the presence of Toxins A&B of Clostridium difficile in human stool. It is part of a test called Stool Occult test. This test is used to aid physicians in diagnosing C. difficile-related diseases.

Alrighty, I'm done =). We've got 10 weeks more to goooooooo! Have fun all!

The cutest Lab Freak,
Sharifah

MCT techniques and a the machines stuff..

Hmm..okie..it's my turn again after Nat..this time I'm going to talk more about my MP.

Basically, my MP is about the protein analysis of lung cancer cells that are treated with herb using LC-MS/MS. It mainly involves MCT techniques and chemical engineering stuff such as the machines used. Prior to using the machines, I have to seed cells and here's where all the aseptic techniques and cell counting comes in. Using a hemocytometer, the cells are counted and the concentration of the cells are calculated. This had been elaborated in Najib's entry, so take a look if u guys don't know how to alright! A cell densitiy of 7million cells are seeded and 3 of the culture dishes are treated with 2mg/ml of herb extract after a 24 hr incubation time. Why 2mg/ml then? well, this concentration had been verified by a senior that it is the optimal concentration that will cause a lowest cell viability. An incubation time is required to allow the cells to adhere to the surface of the culture dish and also to proliferate somemore! After that, the cells are killed!! M-PER is added to allow the cell lysis so that protein extraction can be done using a cell scraper. The supernatant is then taken out and stored at -80degrees after a centrifuge step.

Now comes the part that is rather foreign to us, as it's rather chem eng based. 2D-LC is run! Before this, the proteins are trypsin digested and iTRAQ labeled. iTRAQ reagents will label proteins based on the different mass, namely 114,115,116,117. These reagents binds to the peptides (i.e. proteins after the trypsin digestion) at the N terminal of the lysine chain. This labeling will allow the quantification of the different proteins identified. In addition, it allows up to 4 samples to be mixed and run together, therefore lowering the time needed for the run to be carried out! This step then makes my life easier, because i will know if the protein is being up or down regulated based on the iTRAQ ratio that is given.

Ok! back to 2D-LC. 2D-LC is actually a fractionation step to fractionate the peptides samples. This involves 2 column modes, column 1 and column 2 mode and 2 different pumps are also involved, the quaternary pump and the capillary pump. In column 1 mode, the quat pump is the busy one, it pumps the sample that is injected into the Strong Cation Exchange (SCX) column then to the enrichment column and lastly to the waste. The peptides will then be stuck at the SCX column. In column 2 mode, the cap pump becomes the busy one. It pumps the peptides that is stuck at the enrichment column into the RP column. Erm, as some of the peptides will be still stuck at the SCX column, salt of different molarity is run. These salts help to elute the stuck peptides that is at the SCX column into the enrichment column. The irritating thing about this machine is that it lacks a auto fractionator, which means that I'm the fractionator. The eluted samples are collected at a 2mins interval. Which means that at every 2min, I have to change the tube to a new eppendorf tube. Imagine sitting at the lab for straight 4 hrs!! Can't leave the lab at all. Sickening rite!

Now here comes MALDI. MALDI helps in the protein detection and the protein will then be identified by the MASCOT system. I will not be elaborating on MALDI as Sharhirah had already talked about.. so guys..refer there! :)

Okie..thats about all..Just a brief idea on what my MP is actually about and the stuff involve.. If there's any questions, just post them k..I'll answer them asap!

Have Fun people! Back to school soon..haha..

Charmaine
0503186I

back to me agn (:

ouh. when i am blogging, it means that 2months of sip have passed!
how time flies eh?

Ha. Anyway back to serious business.
Yes today i shall post bout the experience i had and what i've learnt in the Mircobiology lab.
A short brief one that is. (=
Basically the microlab consists of 5 benches; namely,
1)Specimen Processing Bench; whereby all the specimens are being processed and sorted out.
2)Urine Bench; whereby necessary interpretation of primary urine culture plates(that are cultured the day before) and performing of tests for all positive tests.
3)Microscopy Bench; its whereby the staff in-charge process specimens such as sputum for diagnosis of TB/myobacteria infection.
4)Blood Bench; its the same process as in the urine bench, just that the specimen is of blood.
5)Miscellanous Bench; as its name called, its of interpretation and culturing of other specimens than blood and urine.

I shall focus more on the Bacteria ID Identification procedure. Its rather interesting, the best part i liked! and its bascially like the die-die-must-know thing in a mircolab. lol

First, gram-staining is done on the unknown bacteria.
Then by using the microscopy lens, identify the bact is of a gram positive (violet colour) or a gram-neg(pink colour).

For the gram-pos bacteria,
a Catalase test is done.
If its catalase positive- it is a Staphylococcus bacteria.
A Coagulate (latex) test is done to further identify its type.
So if its catalase pos, it is either a Stap.aureus or MRSA.
If its a catalase neg, it will be reported as STCH (normal commensal flora).
For catalase neg- it is a Streptococcal organism. Further tests e.g. alpha test, beta-grouping test etc are being carried out. Normally, the beta-grouping method is the most common, it tell us what group type it belongs to.

For gram-neg bacteria,
we'll carry out the biochemical tests (i.e. 7tubes of 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.
Biochemical Tests are of the followings:
1)KIA slant
2)Simmons citrate
3)Urease
4)Motility (& OF) test
5)Indole
6)Phenylalanine slant
7)Malonate test

So, by identifying the positive or negative results of the various tests will help us to differeniate which Enterobacteriaceae group it belongs to!

okay i am done with all the brief short explaination.
If you guys need futher explaination on the principles and the result interpretation of the various biochemical tests, just give me a message! i'll post it asap okay.

Have fun SIP-ing guys/babes. (:


signed,
Natalie

HOT MCT Techniques.

Elo Elo eh eh eh. Haha.

Hey guys wassap. Its been 7 cool/hot weeks since sip started. Its great working in the department of experimental surgery. Its cool looking at the animals but i am doing only invitro work, therefore i do not handle the animals.

In this post i will cover alot on mammalian cell technology stuff as this is the bulk of my experiments. Hope this will also freshen your memory.

Now lets got to the basics which is subculturing of cells. As charmaine mention earlier subculture is done when the cells have reached confluence. In my lab i will subculture the cancer lines (eg. Hep G2) when it is 80-90% confluent. Let my give 2 major importance of subculturing.

1) To prevent overcrowding of cells. Therefore porvides space for the cells to grow as over crowding will result in scenescence of the cells.

2)Subculturing replenish the nutrients contained in the media. Metabolic wastes which could be harmful to the cells at high concentrations are removed during subculturation.

If the cells have not reached confluence, we have to change their media about once or twice a week depending on the cell type. Certain cell lines are slow proliferators while others grows very fast. These slow proliferators will eventually use up all the nutrients and produce waste therefore a media change is needed for them.

Here are the steps to perform subculture. It is different from what we learnt during MCT lessons.

A) Subculturing of Cells (perform in Biosafety cabinet)
1. Discard spent media from culture flask into waste bottle
2. Wash with 10mL PBS and discard
3. Add 2mL of trypsin and incubate for 2- 3 minutes
4. Observe under the inverted microscope if cells are detached
5. After cells are detached, add 8mL of media to deactivate the trypsin and mix well
6. Transfer 10mL of cell suspension into a 50mL centrifuge tube
7. Centrifuge the cell suspension at 1500 rpm for 3 minutes
8. Discard supernatant and resuspend cell pallet while washing with 10mL PBS
9. Centrifuge at 1500 rpm for 3 minutes
10. Discard supernatant
11. For a cell concentration ratio of 1:20, add 10mL of media and resuspend the pallet
12. Prepare a new culture flask adding 19.5mL media
13. Aliquot 0.5mL of cells into new 75cm2 culture flask containing media
14. Check under the inverted microscope for cells
15. Incubate cells in the 370C, 5% CO2 incubator

The difference is in Step no. 6 onwards.

In my lab, after deactivating the trypsinized cells, there is one additional stet which is the washing with PBS (step no. 8). This is done so as to remove as much trypsin as possible. That is why in our MCT lab sessions, some of us had a hard time detaching the cells with trypsin as previously, the trypsin is not fully removed by an additional washing step. The cells are adapting to the enzyme trypsin effect and thus it takes a longer time for the trypsin to work for subsequent trypsinization. Also in my lab, culture flask are changed every time subculturing is done. I guess our school do not do such as it is too costly and it is not for something of high importance such as research. Changing flask regularly helps prevents contamination as older flasks are exposed to the environment which contains many microbes.

Now let me talk about our favourite hemocytometer. Guess what it is used for? Anyone?

Correct! It is used for cell counting. For mammalian cells, cell counting is done for many reasons. Examples are when freezing cells, performing MTT assay and many more. For me i had to perform cell counting as I need to do MTT assay.

For cell counting, I obtain the cell number estimate by counting big(1mm by 1mm) squares at the 4 corners of the counting chamber.

*Sorry ppl im unable to put a nice/hot photoshoot of the counting chamber cos blogger is being a pain for now.*

Trypan blue is used in cell counting to differentiate the dead cells from the living ones. The dead cells will be stained blue whereas viable cells will look white under the microcope. Formula for cell concentration:

C=n x 10^4 x df

Whereby,

c=cell concentration (cells/ml)

n=average no. of cells/mm^2 (average of the 4 squares)

10^4=volume counted

df=dilution factor

Cell counting is crucial in MTT assay as seeding of cells in each of the wells of the 96-well plate must contain equal amounts of cells so that accurate results will be produced when the assay is performed.

Ok guys my post ends here and pls do ask any questions if you are in doubt. Have fun and hope you guys learn alot during SIP. Take care.

Najib Bin Hamid (0503217B)
TG01

Hi everyone! Sorry for the delay in my post there was a bit mixed up in my remembering of the weeks.

I am working in the food and water microbiology lab now. In the lab, the main testing that we were instructed to focus on is on the testing of food samples. Ying Ying has roughly described of routine testing of food and the basics of food tesing principles. Now I am going to touch on the testing of specific pathogen, Salmonella. The most common testing for Salmonella in our lab is the testing of chocolate samples from overseas companies.

For Salmonella testing of chocolate sample:

25 grams of chocolate sample is being weighed and 225ml of skim milk is being added to the chocolate sample. For normal Salmonella testing, buffered peptone broth is being added instead of skim milk. However, as this is a request from the company overseas, we have to follow their instruction as that is their way of testing there. After the addition of skim milk, the food is being homogenized by the stomacher. The stomacher has been described in Ying Ying’s blog posting so I shall not talk about it anymore. After stomaching at 230rpm for 30s, phosphate buffer is then added to the homogenate. After that, the chocolate homogenate is then taken to the incubator for incubation at 37◦C.

After incubation for 24 hours, 1ml of the chocolate homogenate is being pipetted into Tetrathionate Brilliant-green Bile Enrichment Broth (TTB) and 0.1ml is pipetted into Rappaport and Vassiliadis (RV Broth). This is a selective enrichment step to improve the growth of Salmonella. For TTB, 0.2ml of iodine and 0.01ml of brilliant green is added before the chocolate sample is added.

For TTB (incubate at 35◦C):
- Only organisms which possess the enzyme tetrathionate reductase can grow (Only Proteus and Salmonella)
- Proteus can be inhibited by adjusting the pH to about 6.5 or the addition of novobiocin.
- Bile salts promote the growth of enteric bacteria and inhibit the growth of bacteria that do not normally live in the intestine.
- Brilliant green inhibits gram-positive bacterial flora.
For RV Broth (incubate at 42◦C):
- Allows only Salmonella growth by exploiting the following characteristics of Salmonella:
o Ability to survive at high osmostic pressure
o Multiply at low pH values (pH: 5.5)
o More resistant to malachite green (increase amounts)
o Less demanding nutritional requirements

After incubation for 1 day at their respective incubators, the samples from both the TTB and RV Broth is streaked onto Hektoen Enteric (HE agar), Bismuth Sulfite (BS agar) and Xylose Lysine Desoxycholate (XLD agar). 3 different types of agar plates are used as each of them supports the growth of different Salmonella species so any form of Salmonella can be detected.

Mode of action:
Hektoen Enteric agar
- Contains bromothymol blue and acidic fuchsin indicators. Differentiates lactose-positive colonies from lactose-negative colonies.
- Additional carbohydrates (sucrose and salicin) helps early detection of slow lactose-fermenting organisms to prevent false-postive results.
- Thiosulphate and ferric citrate causes H2S positive colonies to become black in colour.
- Blue green colonies with black centres would indicate presence of Salmonella..

Bismuth Sulfite agar
- Selective media for isolation of salmonella species.
- Bismuth Sulfite and Brilliant Green act together as a selective agent by suppressing the growth of coliforms.
- H2S production by salmonella due to presence of sulphur.
- Reduction of bismuth ions forms metallic lusture around the colonies.
- Black centre, light edges surrounded by a black precipitate with metallic luster would indicate presence of Salmonella.

Xylose Lysine Desoxycholate agar
- Degradation of xylose, lactose and sucrose to acid causes phenol red indicator to change to yellow.
- Decarboxylation of lysine by salmonella species alters pH to alkaline causes purple colouration around the colonies.
- H2S production indicated by thiosulfate and iron(III) salt forms a precipitate of black iron sulfide in the colonies.
- Red colonies, translucent, sometimes with a black centre would indicate presence of Salmonella.


If there are any suspicious colonies, biochemical tests should be carried out.
The following descriptions below are the biochemical tests for the confirmation of Salmonella.

Triple Sugar Iron (TSI) differentiates gram-negative enteric bacilli based on carbohydrate fermentation and production of hydrogen sulphide. TSI contains dextrose, sucrose, lactose, phenol red (detection of carbohydrate fermentation) and ferrous ammounium sulphate (detection of hydrogen sulphide). The gas produced during carbohydrate fermentation will change the phenol red indicator from red to yellow. Formation of hydrogen sulphide will cause blackening in the butt of the test tube. To facilitate the detection of organism that only ferments dextrose/glucose, the dextrose/glucose concentration is 1/10 the concentration of lactose or sucrose. The acid produced at the slant oxidizes rapidly, causing the medium to remain red or revert to an alkaline pH. However, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. Loosely capping will allow the exchange of gases.

Lysine Iron Agar (LIA) aids the differentiation of enteric bacilli based on the ability to decarboxylase lysine and to produce hydrogen sulphide. LIA contains bromcresol purple indicator (detection of lysine decarboxylase production), lysine (detection of enzymes lysine decarboxylase and lysine deaminase), ferric ammonium citrate and sodium thiosulfate (both are indicators of hydrogen sulphide). The gas produced during carbohydrate fermentation will cause the bromcresol purple indicator to remain purple. Formation of hydrogen sulphide will cause blackening of the medium due to production of ferrous sulfides. Production of lysine decarboxylase would cause an alkaline reaction (purple). Deamination of lysine would cause an acid slant (red).

Urease Broth is used in parallel with TSI and LIA. Used for the detection of Proteus organism as it causes hydrolysis of urea (colour change to pink).

Indole test is used in the differentiation of genera and species. Tryptone water is used due to its high content of tryptophan. Any microorganism with the enzyme Tryptophanase will cleave tryptophan to produce indole and other products. Kovac’s reagent will then react with indole to form a red ring. False negative results will be produced if the pH falls below (7.4-7.8).

Methyl Red test is used to test the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Organisms that form large amounts of acid from glucose fermentation causes the pH to fall below 4.4 (colour becomes red).

Voges Proskauer (VP) test is used to detect the production of acetylmethylcarbinol (acetoin) from the fermentation of glucose/dextrose. If positive, acetoin is oxidized in the presence of oxygen and potassium hydroxide to diacetyl which reacts with peptone water to give off a red colour.

Decarboxylase media are used in differentiation of gram-negative enteric bacilli based on the production of arginine dihydrolase and lysine and ornithine decarboxylase. When dextrose is being fermented, the bromocresol purple indicator changes from purple to yellow. The acidic condition also stimulates decarboxylase activity. When the amino acid is degraded, a corresponding amine is being produced. The amine then elevates the pH of the medium, causing a colour change of the indicator from yellow to purple.

Simmon’s Citrate agar is used for the differentiation of gram-negative enteric bacteria on the basis of critrate utilization. Organisms that are able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole source of nitrogen and carbon will produce an alkaline reaction by changing the bromthymol blue indicator from green to blue (alkaline).


After incubation, if the biochemical tests results are as follow, the chocolate sample can be presumed to contain Salmonella:

TSI (incubate at 35◦C for 24hours): Acid butt (yellow), alkaline slant (red) with H2S production (black)
LIA (incubate at 35◦C for 24hours): Alkaline reaction (purple) with H2S production (black)
Urease test: Negative (no colour change)
Indole test (Tryptone water + Kovac’s reagent): Negative
Methyl Red test: Positive (distinct red colour)
VP test: Negative (no colour change)
Base decarboxylase: Negative (yellow)
Glucose/Xylose decarboxylase: Negative (yellow)
Lysine decarbosylase: Positive (purple)
Ornithine decarboxylase: Positive (purple)
Simmon’s Citrate: Blue
Gram staining: Gram-negative bacilli

After that, serological test is being carried out. Each presumptive Salmonella isolate (using TSI agar slant capture) is being tested against Polyvalent O (somatic) and Polyvalent H (flagella, phase I and II) antisera. For both Polyvalents, there is agglutination. This shows that the presumptive Salmonella can be confirmed as Salmonella.

Jeremy Lee
TG01
0503168G