Week 5: Investigation lab (Microbiology)

Oh my, 5 weeks of our attachment have already passed. I'm have also moved onto another lab. From the central processing area (CPA), I'm now in the investigation lab (IV lab). In CPA, we 'prepare' the patients' specimens for testing. Then, these prepared specimens will be sent to the IV lab so that the bacteria present can be identified, so that a diagnosis can be obtained. Afterwhich the appropriate treatment can be given.

Having said so, it also means that the IV lab plays an important role as to whether the patient will receive the correct treatment. So as interns, we are not given much investigation work to do. Instead, we are being tasked with simpler and 'save-able' jobs. 'Save-able' as in if we do anything wrong, they can repeat the test again.

So far, I have done these few things:

- Subculture cooked meat and brain heart infusion broth (BHIB)

- Perform latex agglutination for the identification of Staphylococcus aureus

- Perform antibiotic susceptibility testing for urine cultures.

Subculture of cooked meat and BHIB

Cooked meat is an enriched media used to cultivate anaerobic bacteria. But according to my mentor there, cooked meat is also used as back-up. In some cases, there may be too little bacteria present in the specimen to show up on the agar plates. As the specimens are submerged into the cooked meat media, any bacteria present cannot 'escape'. After incubating the cooked meat for 1 day at 35°, the media will be subcultured onto blood agar and anaerobic plates. Blood agar is used as most bacteria can grow on blood agar. Anaerobic plates are used to detect the presence of any anaerobic bacteria.

Cooked meat medium [1]

BHIB is a general-purpose medium normally used to cultivate fastidious, non-fastidious, aerobic and anaerobic bacteria. But this BHIB has added antibiotics, oxacillin, to detect methcillin-resistant S. aureus (MRSA). So all bacteria, excluding MRSA, sensitive to oxacillin will be killed. MRSA will not be killed by oxacillin as it is resistant to oxacillin. So this type of BHIB is used for MRSA screening. The BHIB will be incubated for 1 day at 35°, and after 1 day, the broth will also have to be subcultured onto MRSA plates.

MRSA plates are actually made up of either blood agar or mannitol salt agar with added methicillin or oxacillin. It is selective media used to detect MRSA. Presence of MRSA can be indicated by purple colonies present on the agar.

MRSA growing on MRSA plate [2]

Latex agglutination for identification of S. aureus.

This test works by detecting 3 components:
- Fibrinogen affinity factor, also known as bound coagulase or ‘clumping factor’
- Protein A, which has an affinity for crystallisable fragment (Fc) of the gamma

immunoglobulins (IgG)

- Capsular polysaccharides of S. aureus

The reagent used in this test contains latex particles that have been sensitized with fibrinogen, IgG and specific monoclonal antibodies specific against capsular polysaccharides of S. aureus. A combination of these 3 components allows both highly and poorly encapsulated strains of S. aureus to be recognized

For highly encapsulated strains of S. aureus, antibodies specific to the capsular polysaccharide will be used to agglutinate the bacteria. For poorly encapsulated strains of S. aureus, the fibrinogen and IgG will be used to agglutinate the bacteria.

Positive result = agglutination observed with loss of'red background

Negative result = no agglutination with no loss of red background

False-positive results may occur when there is a cross-reaction with Streptococcus spp. as some strains of streptococcus contains a protein with an affinity for the Fc portion of the IgG. So it may react with the latex reagent, thus causing a false positive result.

False-negative results may also occur as some S. aureus do not produce fibrinogen affinity factor (clumping factor), protein A, or capsular polysaccharides. Without the capsular polysaccharides, no specific antibiotics will produced.

Antibiotics susceptibility testing

This form of testing is used as a guide to the doctors as to which type of antibiotics would work best to kill the bacteria present in the patient. So tests are performed in vitro and how well the organism grows in response to the antibiotics will be measured. [6] The antibiotics susceptibility testing is normally done using Mueller-Hinton agar. Mueller-Hinton agar is used for this test because of a few reasons:
- Good reproducibility of the medium
- Simplicity of its formula
- A lot of experimental data are available regarding the usage of this medium [7]

Type of antibiotics to be used for the test is determined by a few factors:
- Target location of the antibiotics (e.g Antibiotics meant to treat bacteria in the brain

must be able to pass through the blood brain barrier)

- Target bacteria, as some antibiotics work better for gram-positive bacteria, while other

antibiotics work better for gram-negative bacteria)
- Avaliability of the antibiotics, as some antibiotics may be more commonly used than others.

As mentioned above, different types of bacteria will require different groups of antibiotics to be used. The groups of antibiotics are described as ‘lines’.

Staphylococcus = A line

Streptoccus = E line

Enterococcus = BE line

Pseudomonas aeruginosa = E line

*Enterobacteriaceae = F line

*Enterbacteriaceae refers to any other bacteria not mentioned in the previous lines. They include Escherichia coli, general Pseudomonas and so on.


Procedures
1. Inoculate single colonies of bacteria into saline. The turbidity of the mixture should match

the 0.5 MacFarland turbidity standard.

2. Dip a cotton swab into the mixture and drain off excess mixture.
3. Swab the entire surface of Mueller-Hinton agar with the cotton swab by turning 60 degrees

after each swab.

4. Stamp the appropriate group of antibiotics onto the Mueller-Hinton agar.
5. Incubate plates at 35° for 1 day.

Result

Bacteria susceptible to the antibiotics = zone of inhibition will be observed around the disk.
Bacteria resistant to the antibiotics = zone of inhibition will be absent around the disk.

Antibiotics susceptibility test [3]

So these are basically what I'm doing in the IV lab. I will be starting on biochemical tests next week. Yes, all the urease, oxidase, catalase tests and so on. So wish me luck!

Picture references:

1. http://www.oxoid.com/uk/blue/press/press.asp?art=Y&arch=Y&pRef=PR023605&c=uk&lang=en&yr=2005

2. http://www.chemoquip.com/

3. www.med.unsw.edu.au/pathology-cds/

-Li Ping-

TG 02

Pictures of urine cultures

To Benjamin


For Q1


This is how the urine specimen on blood agar plate looks like after incubation..












(Picture taken in lab. Permission given by supervisor.)



And the urine specimen on CLED plates..













(Picture taken in lab. Permission given by supervisor.)




LeeJin
TG02

Hello I hope that you guys are there are enjoying your SIP at ur individual places. I’m in the clinical biochemistry department for my SIP. And I am going to talk about bilirubin measurment for neonates. In my lab we use a bilirubinometer that is a direct spectrophotometer that measures total bilirubin in neonates( babies ) by using their serum samples. These samples are usually collected in capillary tubes as neonates do not have much blood to be drawn out for analysis.
Mechanics of the bilirubinometer

1. Light passes through the cuvette and is split by a dichroic mirror and directed to 460 and 550 photo detector

2. Output of the photometer is calculated by control electronics to measure total bilirubin

3. bilirubin and oxyhaemoglobin’s peak absorbance is 460nm. It is necessary to measure oxyhaemoglobin at 550nm The difference in reading at 460 and 550nm is the total bilirubin value.

4. usually haemolyzed samples do not affect the result as the bilirubinometer in my lab substracts the oxyhaemoglobin value from the total value that is collected by the photometer .

5. this bilirubinometer only measures neonate sample as interfering substances become more prevalent and will affect results for adults

Interfering substances
Pigments that are absorbed at 460nm will affect the reading of the sample in the bilirubinometer. Some examples are lipochromes and carotenoids. Neonatal samples that are collected usually lack such interfering substances to affect the reading

Certain drugs also interfer with readings, like for example sulfonamides, phenols and amines.

Intravenous fat emulsions and lipaemic samples are also know to affect the reading. Such an example is Lyposin that has affected direct spectrophotometric measurements

Daily Controls
2 assayed galss cuvette is provided by the manufacturer, it is usually done between runs, at the start of each day.

EQC
my lab has taken part in certain EQC programmes for bilirubinometer for neonates. Some examples are CAP, neonatal bilirubin assay programme





Materials
Sample
test tube
special pipette
centrifuge machine
cuvette
bilirubinometer

Methods
1.Place capillary tube sample into test tube affixed with plastic stopper to prevent blood from leaking out during centrifuging. This will cause of loss of serum which is precious as neonates are involved

2.place in centrifuge and counter balance

3.spin for 5 mins at 3500 rpm

4.using a capillary tube pipette, fill cuvette with serum. The cuvette volume is 20 uL but there’s no need to measure the sample as the exact volume is determined by the cuvette. Cuvette needs to filled full.

5.the outside surface of the cuvette must be free of serum, dust and smudges as it will affect the machine’s capability of interpreting the result

6.bubbles will affect the reading, thus the rectangular area of the cuvette must be cleared of bubbles. Tap lighting on cuvette to remove bubbles.

7.place cuvette in bilirubinometer and press start button.

Yuxuan

picture reference: www.reichertai.com

Week 4 - Virology

Hi everyone, I am attached to the tissue culture section under virology department. Basically, this section is an area that is responsible for the growth of cells, preparation of media and also the subculturing and maintenance of cells. It is something like what we do in mammalian cell technology- in which aseptic techniques is very important and contamination is not allowed!

Virus needs living cells to grow; hence in order to isolate and grow these viruses we need different types of cell culture. In this section, we will prepare 11 types of cell lines, as there is no single cell line that can grow all the virus. The examples are as follows:

Cell line	Type of virus that is grow in
Hela (cervical cancer cell)	Adenovirus, rhinovirus, polio virus
Hep (throat cancer cell)	Respiratory syncytial virus, polio virus
Hel (human embryo lung fibroblast)	Herpes, varicella, cytomegalovirus
Vero/slam (genetic engineered cell from vero)	Measles virus
Vero (African green monkey kidney cell)	Adenovirus, enterovirus, SARs virus
L20B (mouse cell)	Polio virus
C636 (mosquito cell)	Dengue virus
LLC (African monkey kidney cell)	Influenza virus
MDCK (Dog kidney cell)	Influenza virus
ML (mink's lung cell)	Herpes virus
RD (muscle cancer cell)	Enterovirus

The things that i have learnt so far are as follows. Firstly, Let's start from media preparation.

Minimum essential medium (MEM) preparation

1. Add the consituents (NAHCO3, water, hepes and MEM powder) in their respectives volumes and amount into a sterile flask.
2. Mix well by placing a magnetic stirrer into the flask and place it on a hotplate.
3. Switch on the hotplate to stir the contents for 1 to 2 minutes.
4. Sterilize the media by using peristaltic pumps (0.2micrometer pore size membrane).
5. Filter the media through filter membrane into different sterile bottles inside the BSC II ( biological safety cabinent class two).

Sterility test for the media prepared ( from above):
- Add 1ml of the media prepared to both nutrient and sabouraud broth respectively.
Nutrient broth is to test for growth of bactera while sabouraud is for fungus growth. This serves to check that there is no contamination in the media ( indicated by turbidity) after 1 -2 weeks before the media is used to grow cells.

Next, retrieving the cells from liquid nitrogen.

Cell recovery from liquid nitrogen

1.Check for the type and location of cells that sholud be taken out.
2. Put on cryogenic gloves and face shield.
3.Take the respective cryotube out.
4. Thaw the cryotube quickly in 37 degree waterbath.
5. Disinfect the outside of the cryotube in 70% alcohol.
6. Pipette the thawed cells into a culture flask containing 5ml of fresh growth media.
7. Change the media the next day.( as it contains DMSO- cryoprotectant, that is added to the cells during freezing. It serves to lower freezing point and minimize the effect caused by freezing )
8. Alternatively, centrifuge the thawed cells to obtain the pellet (discard the supernatant).
9. Transfer the pellet into fresh growth media in culture flask. ( In this case, there is no need to change the media the next day as DMSO have been removed.)

Lastly, storage of cells in cell bank.

Freezing of cells for storage
[ Cells to be stored in cell bank must achieve the standard concentration of 5 x 10^6 cells/ml]

1. Trypsinise the cells that are to be freezed ( Wash 2 times with Phosphate buffered saline and then add trypsin solution)
2. Prepare the freezing media(0.3ml DMSO and 2.7ml fresh MEM) and prechill in fridge.
3.Add cells to media in a plastic centrifuge tube.
4. Take 1 drop from the centrifuge tube and place in on a parafilm.
5. Add a drop of trypan blue to this drop and mix well - 1 drop : 1 drop, so gives dilution factor of 2. ( Trypan blue will stain the dead cells , thus allowing the viable cells to be quanitified)
6. Load it into the hemocytometer
7. Count the number of cells under microscope.( count 4 squares then take avarage and mutiply by dilution factor)
8. Determine the volume of cells to be loaded into the cryotube by microsoft access. ( Consist of a formula that is already set up for calculation of the volume needed to load in cryotube - to achieve the standard concentration of cells)
9.Add the desired volume to the cryotube and seal it with masking tape parafilm.
10. Place the cryotube in Mr frosty ( Freezing container with isopropanol - to allow the cryotube to come to temperature of freezer at about 1 degree/minute)
11. Place Mr frosty in -85 degrees freezer before transfering it to liquid nitrogen.

That's all for now. I will be moving on to virus isolation section soon. Hopefully, i can share with you guys more interesting things.


For more information on the viruses above, you may want to visit these websites:
http://www.emedicine.com/med/byname/adenoviruses.htm
http://www.emedicine.com/ped/topic2707.htm
http://www.dhpe.org/infect/rsv.html
http://www.dhpe.org/infect/cytomegalo.html
http://pathmicro.med.sc.edu/virol/picorna.htm

Goh Shihui
0607135A
TG02

Week 3 – Medical Microbiology

Topic: Urine and Stool cultures

Hi guys I’m attached to Microbiology department for 20 weeks. This department consists of various labs. So for the first 5 weeks, I will be in Central Processing Lab. This lab processes the specimens upon receiving it. Wanna share with you how we process urine and stool specimens.

Urine Culture

Principles:
- Urine culture is done to detect bacteriuria.
- Urine is cultured onto the media by surface streaking. It will then be incubated to promote the bacterial growth and obtain single colonies and allow viable count.
- It helps in the diagnosis of Urinary Tract Infection (UTI).
- Primary cause of UTI: members of Enterobacteriaceae family. Some possible causing agents are Escherichia coli, Proteus spp. and Klebsiella spp.
- Fresh midstream urine should be used.
- Specimens come in a form of urine bottle or Dip slide container.

Materials:
1. Patients’ urine specimens
2. BAP & CLED plates
3. Dip slides

Picture taken from http://www.solarbiologicals.com/images/Image4.jpg

4. Disposable inoculating loops (1µl and 10µl)

Picture taken from http://canadawide.ca/products/596-300.1.html

5. Disinfectant container , Biohazard waste bin


Procedures:

For specimens in urine bottles:
1. Dip a 1µl disposable inoculating loop vertically into the specimen.

(If the urine specimens come from nephrostomy or collected in the Operating Theatre, an addition of 10µl disposable inoculating loop should be used. So a total of 2 BAP and 2 CLED agar plates are used.)

2. Perform surface streaking onto BAP and CLED agar plates.

Surface streaking
1. Streak a vertical line down the agar plate.
2. Streak closely from the top. Make wider streaks as you move downwards to obtain single colonies.

3. Discard the disposable inoculating loops into a disinfectant container.
4. Discard the specimens into the biohazard waste bin.
5. Incubate all the agar plates at 35°C overnight.

For specimens in dip slides:
1. Incubate it at 35°C overnight after receiving the specimens.


After incubation, all agar plates and dip slides are sent to the Investigation Lab to quantify and identify the colonies if there is any bacterial growth.


Stool Culture

Principles:
- Stool culture is done to identify the cause of infection in the intestine, diarrhea or bloody stool.
- It is commonly used to identify Salmonella, Shigella, Campylobacter and Vibrio spp.
- Diarrhea is usually caused by Vibrio spp. so an addition of Thiosulphate-citrate bile sucrose agar (TCBS) and Alkaline Peptone Water (APW) enrichment broth are used to enhance the growth of this genus.
- Selenite F broth is an enrichment broth for the growth of Salmonella spp.
- Bloody stool is caused by enterohemorrhagic Escherichia coli (EHEC). Commonly by E. coli O157:H7 strain. Sorbitol MAC media is used to differentiate E. coli O157:H7 from other E. coli strain.

Materials
1.	Patients’ stool specimens
2.	Inoculating loops
	For solid stool	For watery stool	For bloody stool
3.	Blood Agar Plate (BAP) MacConkey agar (MAC) Salmonella–Shigella agar (SS) Campylobacter agar Selenite F broth
4.
5.
6.
7.
8.		TCBS	Sorbitol MAC agar
9.		APW
10.	Wooden stick	Cotton swabs

Procedures:

1. Use wooden stick / cotton swab to take up the specimen and streak onto the appropriate agar plates. Then place it into Selenite F broth and/or APW.

2. Use inoculating loops to perform tertiary streak for all agar plates.

For Sorbitol MAC agar, a control is done to ensure that the agar is functioning.)

3. Discard the inoculating loops into a disinfectant container.

4. Incubate all media at appropriate conditions.

Media	Incubation
BAP	35°C , 18 – 24 hours
MAC
SS
TCBS
Selenite F broth	35°C , 12 – 18 hours
APW	35°C , 6 – 12 hours
Sorbitol MAC agar	35°C , 24 hours
Campylobacter agar	42°C , 48 hours with 85% N2, 10% CO2 and 5% O2

5. After incubation, Selenite F broth and APW are sub cultured and then incubate it.

6. After incubation, all the plates are to the Investigation Lab to identify the colonies if there is any bacterial growth.

Posted By:
LeeJin
TG02

To Xin Ni

For the S. aureus:

Some organisms require growth factor X while others may require growth factor V. Some organisms may even need both growth factors. But the blood agar only contains factor X. So S. aureus is needed to provide the factor V. ^[2] S. aureus is also used to detect Haemophilius influenza virus. This is known as satellitism.


For the different medium/ media:

Cooked meat

Enriched media

Amino acids (from meat particles), other nutrients (e.g glucose, hemin and Vitamin K), reducing agents (e.g glutathione)

Favours the growth of anaerobes [4]

Growth in medium = turbid medium or gas bubbles in medium. Blackened and disintegration of meat particles = proteolysis occurred.

GC plates	Selective medium	1.Peptone mixture 2. Corn starch 3. Buffering system	To isolate Neisseria gonorrhoeae 2. Absorbs toxic metabolites 3. Maintain neutral pH[6]	If N. gonorrhoeae is present, colonies can be observed on the plates.
LIM broth	Selective enrichment media	Todd Hewitt broth*, yeast extract, colistin, nalidixic acid.	Detect presence of Group B streptococci (e.g Streptococcus agalactiae), especially on specimens from the genital area. [7] Yeast extract is used to enhance growth of group B streptococci.	The broth will be subcultured on blood agar plate and incubated at 5% CO2. If the bacteria are present, colonies can be observed on the plates.

* Todd Hewitt broth consists of: Heart infusion from solids, peptonen, dextrose, sodium chloride, disodium phosphate and sodium carbonate.

Hope it helps =).

-Li Ping-

Haematology

Topic: Different tests done in Routine Haematology

There are many sections in the Department of Haematology. The sections include Routine Haematology, Coagulation, Hb Electrophoresis, bone marrow, flow cytometry, etc.

For the first 4 weeks, I am attached to the Routine Haematology section. There are many tests done in the Routine Haematology lab. Examples of such tests includes ESR, malarial parasite, Hb H inclusion bodies (Thalassemia), reticulocyte count, CBC, differential count, platelet count, body fluid count, etc. In the lab here, ADVIA120 is used for FBC which icludes CBC, differential count, platelet count and reticulocyte count. Reticulocyte count can also be done manually; however, it only applies to neonatal blood.

For this entry, I shall share with all of you more about Erythrocyte Sedimentation Rate (ESR). I will also touch on malarial parasite test, thalassemia and manual retic test.

ESR

For ESR, a circle is indicated on the cap of the EDTA tube during processing at the reception.

Principle

ESR measures the rate of erythrocytes settling in human plasma placed vertically over a time of 1 hour. The value, which is the distance from the bottom of the plasma meniscus to the top of the erythrocyte sediment, is then read off from the pipette in millimeters (mm).

Procedure

1) Check the patient’s particulars on the request form and specimen.
2) Label the pre-filled vial (ESR dilution vial) with the specimen number and record the specimen number into the record book.
3) Use an applicator stick to check for presence of small clots.
4) Mix the blood in the EDTA tube by inverting the tube gently for a minimum of 12 times.
5) Remove the stopper of the vial and pipette 800uL of EDTA blood into the pre-filled vial up to the marking on the vial.
6) Replace the stopper and invert the vial for about 5 to 6 times.
7) Insert the ESR graduated pipette through the pierceable stopper by supporting the pipette and stopper with one hand and use the other hand to hit the pipette down. Then, use both hands to push down the pipette until it touches the bottom of the vial.
8) Allow pipette to stand for exactly 1 hour. Set timer and record the time for reading (1 hour after you set the test) into the record book together with your initials.
9) At 60+/-1 min, record the distance, in mm.
10) Record the numerical values into the record book and enter result in the Meditech system (LIS) with initials. If results are greater than 140 mm/hr, it is reported as >140 mm/hr.


Range: Male (1 – 10 mm/hr)
Female, up to age 50 (3-15 mm/hr)
age 51 and above (3-20 mm/hr)


Factors affecting ESR:
- Erythrocyte-plasma ratio
- Bore of the tube
- Hct
- Verticality of sedimentation tube
- Dilution of blood sample.
- ESR also depends on the ability of erythrocytes to form rouleaux. If there is an increased in the concentration of plasma proteins (mainly fibrinogen), erythrocytes are able to form rouleaux thus there will be an increase in ESR.


Malarial Parasite

To test for malarial parasites:
Prepare 2 thick and 1 thin blood films.

Thick films are prepared by using 2 applicator sticks to drop the blood onto the slide and spread to the size of a 10cent coin. It is best to lyse the RBCs so that the malarial parasites can leak out from the RBCs if there is any. The thick films are then air-dried at room temperature before staining it with diluted Giemsa stain for about 15mins. Diluted Giemsa stain is made up of 9 parts of pH7.2 buffer and 1 part of Giemsa stain. After 15mins, the slides are then rinsed with pH7.2 buffer.

For preparation of thin films, it is the same as doing the normal blood smear using the spreader. The thin films are then stained with Leishman’s or Wright’s stain for 7mins or placed into the autostainer.

NOTE: Thick films should not be dried in 37 degrees incubator or exposed to heat.

Thalassemia

Add the same amount of stain (1% brilliant cresyl blue) and blood into the test tube. Incubate in the water bath for 1 hour. Use paraffin to cover the tube to prevent evaporation. After 1 hour, do a blood smear.


Manual Retic

Manual retic is done only for neonatal blood or when ADVIA120 (the machine we are using) shows an asterisk under the value for retic count.

It is done in almost the same way as thalassemia. The same amount of stain (1% brilliant cresyl blue) and blood into the test tube. Incubate in the water bath for 15 mins. After 15 mins, a blood smear is done.

________________________

All the smears/blood films for FBC, malarial parasite, thalassemia and retic done are sent to the screener to be examined under the microscope.

________________________

POSTED BY:
Malerie
TG 02