micro lab sheet # 5-Dania Salhab

Microbiology lab #5
Week number (7)
Date of the lab: 1-2/11/2010
Done by:Dania Salhab

Urine Culture

The doctor started the session by giving us table that is entitled with (Tentative differentiation of commonly isolated clinical aerobic enteric bacilli by means of Kligler's iron agar and other biochemical tests), this means that we want to do a biochemical tests in order to test Enterobacteriaceae. As we can see in the table, we have two groups:
1) Organisms those are isolated from urine, which are the upper four organisms.
2) Organisms those are isolated from stool, which are the rest except Acinetocter which is isolated from blood.

NOTE…
There are some fluids in our body like urine, CSF, blood and the peritoneal fluid that should be sterile in normal people but the stool is not.

Our lab today will be about the first upper four organisms. The sample that we take it from our patient (urine sample) should be taken in the early morning for the dilution of the urine not to happen, and we it take using a sterile cup.

The sample should be cultured as soon as possible for the bacteria not to grow, because we are interested in the number of the cells. It is cultured on blood agar and Macconkey (to detect gram negative bacteria) in percentage of 1/1000-1/100, after that we incubate the plates for 24 hours at 37 degree.

Sometimes after incubation we find an empty plate this means there is no bacterial growth. But if we find colonies we should examine the bacterial growth (heavy, medium or light) by doing a microscopic counting, and:
1) If the bacterial cells are less than 10^5 in number (countable), then the UTI isn't due to a bacterial infection. In this case we ask for another sample, why:
- Maybe there was an error during taking the sample
- The patient is taking antibiotic that may lower the cell's number and he didn't told us, in this case we ask the patient to stop taking it for at least 48 hrs before taking the urine sample.
OR
- The patient is taking other medications that are affecting.
2) If the bacterial cells are uncountable, that means the UTI is caused by bacteria. Now we should know its type in order to give antibiotic, this is done by doing some biochemical tests.

By examining some samples in the lab and comparing it with the table we have, we saw that E.coli's colonies have a deep pink color because it is a strong lactose fermentor (changing the original orange color of the Macconkey), on the other hand Enterobacter's colonies have a red/yellow color because it is a weaker fermentor than E.coli, also produce gas so its colonies have a bad smell. Other organism we saw…Klebsialla, its colonies have a very mucoid appearance and that because this bacteria has a capsule.

NOTE…
- Always one type of bacteria causes UTI.
- The most common cause of UTI is E.coli.

Now after counting, we should do some biochemical tests in order to make sure of the bacterial type; these tests should be done for pure cultures only. We use a loop for transferring the organisms from the Macconkey into the test tubes.
The biochemical tests are:
1- Kligler's iron agar…is red in color, solid and comes as a powder. Then we dissolve it in water (~15ml), put it in test tubes and cover it with cotton. We put these test tubes in the autoclave to make sure they are sterile. After that we should tilt them and till the agar gets solid. This tilting is because we have two types of fermentation:
- Lactose fermentation at the surface of the tube (aerobic bacteria).
- Glucose fermentation at the bottom of the tube (anaerobic bacteria).
… We use this test to show four reactions:
- Glucose fermentation…the butt of the media becomes yellow if the organism is glucose fermentor.
- Lactose fermentation…the slant (الميل/الانحدار) of the media becomes yellow if the organism is lactose fermentor.
- H2S production...the butt of the media becomes completely black.
- Gas production…then a space at the bottom of the test tube will appear.

2- Urease…
- Pink color >> +ve test.
- Yellow color is preserved >> -ve test.
3- Citrate…
- Blue color >> +ve test.
- Green color >> -ve test.

4- SIM agar…we use this colorless media to show three things:
- S >> H2S production, then a black color will appear.
- I >> Indole…
We add an indicator called povax reagent, if it is +ve test like E.coli then a pink ring will be formed.
- M >> Motility, then the media will be turbid.

… As we saw in the lab:
1) E.coli
- Yellow slant >> deep lactose fermentor.
- Yellow butt >> sharp glucose fermentor.
- Empty space at the bottom of the tube >> gas producer.
- No black color at the butt >> doesn’t produce H2S gas.
- Doesn't contain urease enzyme >> yellow color is preserved.
- Turbid media >> motile.
- Pink ring >> indole +ve.

2) Enterobacter and Klebsialla
- Weak yellow slant >> weak lactose fermentor, but in order to differentiate between them we use SIM agar to test the motility:
- Klebsialla is non-motile.
- Enterobacter is often motile.

3) Citrobacter
- Yellow butt >> weak glucose fermentor.
- Yellow slant >> weak lactose fermentor.
- Black butt and bad smell >> H2S producer.
- Blue media >> citrate +ve, (the origin of its name).

The end