Sunday, 1 July 2007

Cheng Hong: Mircobiology

Subject title: Microbiology

Urine culture for diagnosis of Urinary Tract Infection(UTI)

  • To culture patient’s urine sample to look for bacteria growth(mainly E.coli) which is the main cause of UTI
  • Using a 1µl inoculating loop and dip just below the surface of the urine sample and draw a straight streak down the plate(both MacConkey and blood plate), then steak zig-zag lines across the agar(this is to obtain isolated colonies for identification and testing
  • Incubate the plates overnight aerobically
  • Obtain the plate the next day for identification of bacteria colonies
  • If there is growth(pure/mix bacteria growth), look out for E.coli which morphology is white, slightly bigger than other bacteria colonies and has a very “typical smell”
  • Do a indole test on the suspected E.coli colonies: if test result is green(positive), if its pink(negative)
  • If the bacteria is a fermentative Ecoli the MacConkey plate should be reddish(ferment lactose) if it is not(yellow plate), we have to do a MUG test to confirm if it is a E.coli using Bacticard-Ecoli test kit which is bought commercially
  • First, 1drop of rehydrating solution is added, then pick up some of the colonies using a inoculating loop and add to the test card and incubate at RT for 15mins
  • Then add 1 drop of substrate and look under fluorescence light, if there is fluorescence (positive for Ecoli) vice versa
  • At the same time for each patient a Urine FEME(WBC,RBC,Epithelial cell count) is also used to help in the diagnosis
  • If the patient’s WBC count is high(50,000cfu/ml onwards there is a high chance of the patient being diagnosed with UTI if there is Ecoli growth on the agar
  • After reporting the results to the doctors through LIS the culture is sent for verification and sensitivity testing(Antibiotic susceptibility testing)

    Stool culture for diagnosis of cause of diarrhea
  • For diagnosis of diarrhea there are 3 main types of bacteria we are looking for: Shegella, Campylobacter, Salmonella
  • For stool culture there are 3 types of media which is needed to be inoculated on(XLD plate, Campylobacter Plate, Selenite F broth)
  • (Working behind a fume hood) Use a wooden stick to obtain some stool sample and inoculate on the XLD and Campy plates) each plate can have 2 different patient sample)-do streaking
  • Use the remaining stool sample on the stick and put it into the Selenite broth
  • Incubate the plates overnight(XLD, Selenite in 5% CO2 incubator and Campy plates in anaerobic jar with gas pack at 42oC-2days)
  • Obtain the plates for interpretation the next day(XLD plates)
  • If there is growth of black colonies(colonies are not individual, merge with neighboring colonies) we can interpret as growth of salmonella(cause of diarrhea due to ingestion of uncooked/ partially cooked meat)
  • Send the plates for verification and sensitivity testing

    Rotavirus Antigen Identification
  • Pick up patient’s stool sample and inoculate in to 1ml of buffer(comes with the commercial test kit)
  • Gently mix the sample with the buffer
  • Add 4drops of the solution from step 1 into the Rotavirus Quick test kit
  • If only a blue line is seen the patient is negative for rotavirus antigen
  • If a blue and red line is seen, the patient is positive for rotavirus antigen

    VP3 test
  • Vp3 test is done using a vaginal swab to test for Trichomonas, Gardnerella, Candida
  • The swab is placed into a tube and the excess is cut off using a scissors(remember to disinfect with isopropyl alcohol)
  • 12 drops of lysis solution is added and the tube is capped and incubate it in a heating block for 10mins at 85oC
  • Then add 12 drops of buffer and gently flick the tube and cap it with a filter nozzle
  • Add 4 drops of substrate solution to the 7th well of the test kit and dispense the solution from the tube into the first well
  • Then the PAC(test card) into the first well and place the whole kit into the Affirm VP3 machine and run the machine
  • The machine will automatically move the PAC from the first well to the last well
    *those who want the contents of the individual wells please let me know
  • At the end of the run if there is a blue colour on any of the respective organism it shows that the patient have the infection

*When streaking the plate always use 3different sides of the inoculation loop to make the primary, secondary and tertiary streak to be able to get isolated colonies.

*When doing biochem test or other test(eg:indole/oxidase test) use colonies grown on natural media(eg:blood agar)

If there is any question regarding the postings please comment, thanks

*Cheng Hong*


Debra said...

Woah, seems like you had quite alot of stuff to do this week. =)

Anyways, just to clarify, what does XLD (plates) stand for? And what makes the salmonella colonies black in XLD plates? Is it something in the media?

Also, any idea how the rotavirus quick test works, as in the principles? It seems like a ready made test kit. Thanks!
- Debra, TG02

Star team said...

Hey Cheng Hong, I just want to ask you what is a MUG test what is the differences between primary, secondary and tertiary streak? and what do u mena by 3 sides of the loop.

Hope you reply soon

Eugene Wong TG02

VASTYJ said...

Why must the stool samples be subcultured onto the 3 different types of media (XLD plate, Campylobacter Plate, Selenite F broth)? What are their functions?

Andre, TG01

Distinction in Disaster! said...

Hi there, for the stool culture in diagnosing diarrhea cases, you mentioned that the presence of Salmonella is indicated by the growth of black colonies. How about Shegella and Campylobacte?

Charmaine Tan, TG01

VASTYJ said...

hihi.. juz wanted to ask a few qn out of curiosity..

for the quick test for rotavirus antigen identification.. are there any negative or positive controls to be done when doing it?

and the next qn would be.. for vp3 test.. how useful is it in diagnosis of microorganisms infection in the female reproductive system?

my last qn would be for UTI diagnosis.. wad would be done if its negative for the tests.. i mean are there any further investigations that would be performed if the results turned out to be negative (not E.coli)?


Jia Hao

VASTYJ said...

yoyo~! good to see that u have managed to learn much from your 1st week.

okay, Qn time...
1)U mention abt streaking zig-zag lines across the agar, issit the same way as the one we learn?
2)Also ask by eugene, what u mean by using the 3 different sides of the inoculation loop?

yes, just some simple qn... dun wan to ask until so 'cheem' so that ppl can understand better. do check my blog for upcoming new post!

Chaur Lee TG01

J.A.M.M.Y.S said...

Hey seems like you're doing Urine Culture too. Got used to the smell already? Hah.

Anyways, my question to you is this, in my lab, we use Cystine-Lactose-Electrolyte-Deficient (CLED)Agar and Blood Agar Plate (BAP) instead of MacConkey and BAP for Urine Cultures. Can you tell me what are the main differences between MacConkey and CLED?

Azhar TG01

first6weeks said...

Hey! why do u nid to add lysis solution and buffer for VP3 test?
June, TG02

ALsubs said...

hey hi!

Jus want to find out why is that for
1)Urine culture for diagnosis of Urinary Tract Infection(UTI)
2)Stool culture for diagnosis of cause of diarrhea
you have to do an overnight aerobic and anaerobic incubation respectively? Any particular reason?