Microbiology

Hi everybody! Time pssed so fast, SIP is going to end soon!
This week, I am going to share something in the microbiology lab.
Microbiology lab is a place where safety issues are particular important. So I want to talk about the four-biosafety levels.

Biosafety level 1
Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. Work is generally conducted on open bench tops using standard microbiological practices. Laboratory personnel have specific training in the procedures conducted in the laboratory.(Eg: normal routine bench in the microbiology lab)

Biosafety level 2
Biosafety Level 2 is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL-1 in that laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; access to the laboratory is limited when work is being conducted.
Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets.(Eg: the bench working on the respiratory specimen)

Biosafetty level 3
Biosafety Level 3 is applicable to the place where work is done with exotic agents which may cause serious or potentially lethal disease through the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

Biosafety level 4
Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. The laboratory director strictly controls the access to the laboratory. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building.

I want to share one more biochemistry test kit in the micro-lab also. It is PYR rapid colorimetric test.

PYR
PYR is a rapid colorimetric test for the determination of PYRase activity in Streptococci and Citrobacter spp. PYRase activity distinguishes Group A streptococci and enterococci from other streptococcal groups including Group D streptococci.
PYR test kit uses Test Cards impregnated with L-pyroglutamic acid 7-amino-4-methyl-coumarin (7AMC) and dimethylamino-cinnamaldehyde for the detection of PYRase activity. The enzymatic hydrolysis of this substrate by enterococci, Group A streptococci and Citrobacter spp. produces a purple colour followed by the addition of the Developing Solution.

Specimen preparation
When identifying enterococci and Group A streptococci, fresh primary or secondary cultures grown overnight on non selective media such as blood agar give best results. Colonies tested must be Gram-positive cocci and catalase-negative. In case of insufficient growth, a subculture should be performed.When identifying Citrobacter spp. from Salmonella spp. and Enterbacteriaceae, colonies from non-selective media such as (XLD Medium, MLCB Agar, Desoxycholate Citrate Agar, Salmonella Shigella Agar, Brilliant Green Agar or Hektoen Enteric Agar) may be tested. Colonies should be Gram-negative, oxidase-negative and urease-negative.

Test procedure
1. Apply one suspect colony (0.5mm or larger) onto the test area (enough to make a visible smear).

2. Moisten test area with 1 drop of Buffer.

3. Incubate the inoculated Test Card at room temperature (15-30°C) for 5 minutes.

4. Dispense I drop of Developing solution onto the test area. Development of a vivid purple colour on and around the colonies within 20 seconds confirms PYRase activity.

www.labcenter.com.br/novidades.htm

ok that's all~

Enjoy reading:)

Liu Qian

0503935I

Name :Dorene Chen

Department:Microbiology


Hello people,sorry for late blog posting, for this week I will talk about one special type of testing I had during my attachment :Seminal Analysis.


Clinical Significance:

Semen analysis is used to determine male fertility status or to determine the success of a vasectomy.


Semen is composed of spermatoza suspended in seminal fluid, which provides nutrition and volume to transfer the spermatoza to endocervical mucus.


Male in fertility can be affected by a number of causes eg:
1) Decrease in the number of viable sperm
2) Sperm with abnormal morphology
3)Abnormalities of seminal fluid


In determining successfulness of vasectomy, sperm count should decrease slowly from a time span of 6 weeks to 3 months for even longer.


Specimen must be fresh, perferably lesser than 2 hours as viability decreases as time increases. Sample is collected through masturbationm after a minimum of 3-7 days of sexual abstinence and kept into a clean, wide mouthed plastic container and kept at body temperature best.


Method:


Vital Stain (for viability)
1) mix one drop of semen with 2 drops of 1% Eosin Y
2) add 3 drops of 10% Nigrosin solution after 30 seconds and mix
3)place a drop of mixture into a glass slide to make a smear.
4) Viavle spermatoza will be unstained and non viable spermatopza will be stained.( spermatoza shape with purple background seen)
5) Percentage of viabililty can be accessed.


Motility test
1) fill KOVA slide chamber with 20 microlite of neat semen (undiluted, pure specimen)
2) examine under microscope for motility as well as percentage of active, sluggish, non-progressive and non-motile sperms


Sperm count, leucocytes and morphology of sperm

1) specimen is diluted with sodium bicarbonate-formalin solution in 1:20 ratio ( 50 microlite of semen with 950 microlite of solution)
2) fill KOVA slide chamber with 20 microlite of diluted semen suspension
3) examine under microscope for number of spermatoza and leucocytes.
4) access the percentage of normal spermatoza, and abnormal morphology eg: double headed, no head, 2 tails, no tail, wrong head- tail ratio (head too big) etc etc.



Lastly apearance, viscosity, liquefaction time (time taken for semen to be liquid-like), anstinence, time collected, volume of specimen, time received and pH (must be alkaline) should be reported in to seminal analysis as well.


























Counting Spermatoza: chose only 1 large grid.

Source : http://www.clt.astate.edu/wwilliam/cls_1531_seminal_fluid_lab_1_.htm




























undiluted Specimen
Source :http://www2.uni-jena.de/hautkl/elbandro.htm

STUDENT: Cassandrea TG02
LABORATORY: Immunochemistry

The ARCHITECT

o Uses chemiluminescent microparticle immunoassay for the qualitative detection of the antigen or antibody in the serum and plasma of the paitent.

o Two-step immunoassay:
 Sample and antibody/antigen coated with paramagnetic microparticles are combined
 After washing, acridinium-labeled antibody/antigen is added.
 After another round of washing, solutions are added to the reaction mixture, resulting in the chemiluminescent reaction measured by relative light units (RLUs)
 The RLUs is compared to the cutoff signal determined by calibration which will determine if the results are reactive or non-reactive

o Two versions of ARCHITECT in the immunochemistry
ARCHITECT 2000 and ARCHITECT I 2000 SR (newer version)

o Types of test run in ARCHITEST 2000 tests involving hormones like beta-hCG, FSH, LH, Estradiol , prolactin and GH

o Types of test run in ARCHITECT I 2000 SR tests involving viruses like HIV, Syphilis, Hepatitis B and Rubella and thyroid tests which consist of free T4, TSH and calcitonin.

Clinical significance of measuring:

 HBeAg
o First detectable in the early phase of hepatitis B after appearance of hepatitis B surface antigen (HBsAg) early detection of disease
o Both antigen rise rapidly during viral replication in acute infection
o However, patients may not have HBeAg during infection but are still positive for hepatitis B if the antibodies (anti-HBe) to HBeAg are present

 Anti-Hbe
o Used to monitor the convalescence and recovery from hepatitis B infected individuals
o Presence of anti-Hbe and loss of HbeAg after acute infection is a good indication of recovery
o Samples with less than 10.0mIU/mL considered non-reactive
o Samples with greater than or equal to 10.0mIU/mL are considered reactive.

 HbsAg
o Aid in the diagnosis of suspected HBV infection and monitor the status of infected individuals
o Diagnosis of acute or chronic hepatitis HbsAg reactivity should be correlated with patient history and other hepatitis B serological markers
o Negative results need not be tested further but reactive results are retested

 -hCG
o hCG is a sialoglycoprotein initially secreted by the trophoblastic cells of placenta shortly after implantation of fertilized ovum into uterine wall
o Rapid rise in hCG levels after conception makes it an excellent marker for conformation and monitoring of pregnancy
o The hCG increases to peak concentration then decrease an plateau
o HCG levels can be useful in the prediction of spontaneous abortions, aiding in the detection of ectopic pregnancy and multiple gestation

 Ferritin
 High molecular weight iron–containing protein that functions as iron storage compound
 25% of iron in the body is present in various storage forms; 2/3 of the iron stores exist in the form of Ferritin
 Ferritin provides a more sensitive, specific and reliable measurement for determining iron deficiency at an early stage

Hey Guys

Sorry for the late BLogginG. Today i am going to talk about how Amniotic fluid is being set-up.


Amniotic fluid set up

Purpose: The most common reasons for prenatal diagnosis of chromosome disorders of the fetus are advanced maternal age, family history of a chromosome abnormality, abnormal maternal serum screen or fetal defect identified by ultrasonography.

Reason for using amniotic fluid: Amniotic fluid contains cells derived from the amnion, from the gastrointestinal tract and from the skin of the fetus. Some of these cells are viable and are capable of undergoing cell division in vitro. Through amniocentesis, a sample is collected for the purpose of culturing the cells to obtain chromosomes that reflect the fetal karyotype. The in situ culture technique is used where single AF cells are plated onto a sterile glass coverslip in a petri dish where they grow in situ to form discrete colonies.


Steps involved:

1) Usually AF is collected in a tube or sometimes it comes in the syringe.

2) Spin down the tubes containing AF at 1200 rpm for 10 mins

3) Once you have spun down the sample, check the appearance of supernatant and the pellet size

4) After spinning down the tubes, discard the supernatant and dislodge the pellet

5) In one of the tubes, add 1 ml of alpha-AM media and mix it well and in the other tubes add 1 ml of Alpha-bio media. (These are the two media used in my lab for all samples: AF, Chorionic villi etc)

6) There is a need to do 4 cultures. Thus four petri dishes are labeled wit the patients’ name, lab number. (labeled as A,B,C and D)
Note: Each dish has a coverslip attached to the surface of the dish

7) For the dishes A and B, aliquot 0.5 ml of the cell suspension (media used alpha- AM) into each dish

8) As for dishes C and D, 0.5 ml of cell suspension (media used alpha-bio) is added to each dish.

9) Make sure the cell suspension is confined to the coverslips. This is to allow the cell attachment and growth of cells on the surface of the coverslip. (Harvest cells that grow on the coverslipknown as in situ)

10) After which, place the dishes into the respective 37oCCO2 incubator for 4-5days. Culture A and c into incubator A and Culture B and D in incubator B (for backup)

11) And also make sure that you have placed in dishes in the right tray (so that the next day who ever is going to flood the dishes with media knows which dish to flood. )

Diagram to summarize:



Vinodhini
TGO2

Answers to your questions (Sasi's)

Answer to Chaur Lee’s query:

The factors that influence ESR results can be listed under two headings. First, those physical changes in blood plasma and cells, which to some degree the test is measuring and secondly those technical variables that must be avoided when performing the test.

Physical Changes in Blood
The ESR comes about via the interaction between red cells and plasma, therefore any physical variation in either component of blood will affect the result.

The rate of fall is dependent on: - Shape and size (density and surface area) of the red blood cell.Large cells fall faster than small cells. The presence of fewer cells allows for a faster fall rate that increased cell numbers. Cell shape variations , for e.g. abnormal cell morphologies such as sickling (HbS), may slow the rate of sedimentation. Increased albumin slows the fall of cells. An increase in other plasma proteins may increase the rate of fall. Changes in the red cells due to anaemia also usually increases the rate.

- Viscosity of the plasma. This is affected by a number of factors such as concentration of albumin or gamma globulins. Plasma proteins that affect the ESR include the immunoglobulins (IgM) and a number of the acute phase proteins including fibrinogen, C-reactive protein, alpha-1-anti-trypsin, haptoglobin, etc. These acute phase proteins increase with tissue damage (e.g. lupus or rheumatoid arthritis), pregnancy, chronic inflammation and chronic infection and hence increase the ESR rate.

Technical Variables
A number of external factors will affect the result.
Correct mixing of the blood sample before setting up the test is one of the most important factors influencing the ESR result.
Also, the rate of fall is affected by the tube diameter, angle of the tube sides to vertical, temperature, amount of vibrations present, and length of the tube. If a 3o tilt from vertical is present, it is possible that an error factor of 30% may be introduced in the test. A narrow lumen in the ESR tube will slow down the sedimentation rate giving lower results.

Other factors
Females tend to have higher ESR, and menstruation and pregnancy can cause temporary elevations.
Drugs such as dextran, methyldopa (Aldomet), oral contraceptives, penicillamine procainamide, theophylline, and vitamin A can increase ESR, while aspirin, cortisone, and quinine may decrease it.
Age also affects ESR. ESR increases with age in general.


Answer to Charmaint’s query:
Generally, ESR is a marker for inflammation.
It has been also a marker for temporal arteritis and rheumatoid disease as mentioned in my blog. Its also a marker for Lupus.
Apart from that, ESR can also be used as a prognosis marker of ischemic stroke and heart failure.
Many other diseases also increase the ESR: Infection, kidney disease, anemia, diseases involving white blood cells, cancer, and autoimmune and inflammatory diseases.


Answer to Kent’s query:
My lab’s using the Westergren method.
The Westergren method employs a 200 mm, 2.5 mm diameter tube vertically aligned column. The column is filled with blood anticoagulated with EDTA. The distance that the column of blood falls in one hour is recorded and reported in mm/ at the end of 1st hour.
The Wintrobe method employs a shorter tube (100mm) and a different anticoagulant (ammonium oxide and potassium oxalate) is used to mix with the blood.
It has one advantage over the Westergren method as it is able to correct for anaemia while testing patients with marked anemia provides no useful ESR data when using the Westergren method.
Though the Wintrobe method is sensitive for mild elevations, it has a higher false positive rate.
The Westergren method is also more sensitive for changes at elevated levels and may be more useful where the ESR is being used to evaluate the response to therapy for instance, in diseases such as temporal arteritis. Hence, the Wintrobe procedure is not used in most laboratories, the Westergren method being preferred.

Answer to Alex’s query:
Yes Alex, ESR is not diagnostic for any particular disease. It only aids in the diagnosis of the two diseases mentioned in my blod.
The two diseases that Ive mentioned, Temporal arteritis and Polymyalia Rheumatica are basically inflammatory diseases. For instance, ‘Temporal arteritis’ means means "inflammation of the temporal arteries." Since ESR measures inflammation, it is used to aid in the diagnostic aspect of these diseases, to confirm if there is inflammation in the first place. (The ESR rate in these diseases will be always be higher than normal.). When an inflammatory process is present, the high proportion of fibronogen in the blood causes red blood cells to stick to each other. The red cells form stacks called 'rouleaux' which settle faster.
Hence, ESR acts as a marker for these inflammatory diseases.
As for your query on the reference ranges:
The size of the red blood cell affects the ESR reading. The size changes according to the age. For instance, red blood cells are larger in neonates, though smaller in other children. Hence, the reference range is calculated accordingly.

Answer to Elaine’s query:
In our lab, we run control once in the morning daily using the high, normal and low controls which are commercially prepared. We also run another Internal Qc using internally pooled blood (from real patients) three times a day to make sure the precision of the FBC readings amongst the three machines stay constant throughout the day.
And Elaine, guess there was a misinterpretation of what I’ve mentioned about ABO grouping and ESR. Sorry! for not being clear. What I meant was, in our lab, once the EDTA tubes arrive at the haematology department, FBC is run first. Then, ESR is done. Only after these two tests are complete, the respective EDTA tubes are directed to ABO bloodgrouping. ESR is given the next priority after FBC as only then, there is a gurantee that the volume of the sample would be sufficient for ESR. If the respective EDTA tubes happen to go through all the other tests before ESR is run, by the time the tubes reach the ESR section, the volume of blood may not be sufficient for an effective ESR to be run. Hence, I did not mean that ABO bloodgrouping has to be done together with ESR hehe. Hope I’ve cleared your doubts on that.