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*