Tuesday, July 7, 2009

The Urine Bench

Good evening lecturers and fellow course mates. My name is Chu De Ming Jeremy (admin no. 0702919B) and I am attached to Microbiology (Bacteriology) for the whole course of attachment. I will be sharing my experiences over the past 2 weeks in my assigned laboratory. I shall try to keep my posts as clean and concise as possible to ease the monotony.

Before I begin, I apologize for this late post and to make up for this, I will try to share my encounters weekly. Today, I shall start of with my very first week (22nd June - 27th June) in the lab. I title it, The Urine Bench.

The Urine Bench

Urine culture is a very common laboratory test performed to diagnose urinary tract infections (UTIs) which are frequently due to bacterial infections. Escherichia coli, Klebsiella, Enterobacter, Proteus and Pseudomonas sp. are the common culprits of UTIs.
Urine culture requires midstream urine as the specimen because the first bit of urine that passes out may be contaminated from the skin. This is to eliminate possible normal flora growth which can interfere in our identification of pathogenic microorganisms.

In the lab, I was tasked to perform urine cultures for the whole week. Specimens arrived in batches, either in the form of urine, fluid from the kidneys, or dipslides which has already been dipped in urine.

Urine specimen is used to perform aerobic cultures on a split blood agar plate and cysteine lactose-electrolyte-deficient (CLED) media. Direct plating is performed using a disposable 1 micron wired loop and the plates are later sent for incubation at 35°C overnight.
Percutaneous nephrostomy (PCN) is performed on fluid from kidneys. Procedures are similar to aerobic cultures of urine. The only difference is direct plating require the use of both 1 and 10 micron wired loop on 2 separate split blood/CLED agar plates.



A self drawn picture illustrating how streaking is done. Urine is streaked onto blood agar first, then onto the CLED agar. This is to allow trace back if there is any errors or contaminants encountered.




When dipslides are received, they are sent for incubation at 35°C overnight.










Picture of dipslides from http://www.solarbiologicals.com/pages/medical/images/cled-mac-2.JPG
One side is CLED media, the other can be MacConkey (MAC) agar or blood agar.


Blood agar is an enriched differential media that enhances the growth of fastidious microorganism.
CLED media, due to its electrolyte deficient property, prevents Proteus sp. from swarming. It also contains lactose which differentiates lactose fermenters (yellow colonies) from non-lactose fermenters (blue colonies).
MAC agar is a selective differential media that inhibit the growth of most Gram-positive bacteria to stain Gram-negative bacteria for lactose fermentation.

Dipslides are unpopular comp
ared to plating on agar plates as their area is small, making it difficult to make observations. However, one advantage of dipslides is that it can be retained for 2 days before sending it for incubation, as compared to urine where it has to arrive fresh (not more than 24h if kept in refrigerator).

Well, that is all you will need to know for The Urine Bench! Any questions please do not feel hesitant to ask. Thank you.
Check back for the next post in the next couple of days.

Jeremy.

19 comments:

TG01-Group 2 said...

Hello, Chu De Ming! (:

Could you explain what is PCN and the purpose of conducting the test?

Also, by looking at the split blood/CLED agar, how can I determine if there are any contamination encountered?

Thanks!

Siew Ming (:
TG 01 Grp 2
0702862D

MedScientists of Grp 6 said...

Hey Hello!

wanna ask you something! why is urine not streaked onto MacConkey agar? but for the dipslide there is MacConkey agar.are they the same test?

Joanna Yeo
0702054H

low said...
This comment has been removed by the author.
low said...

hello jeremy,

sorry but why a 1micron and 10micron wire loop is used separately on the agar plate ?
whats the difference ?

thanks,
kenneth - Grp 10

TG01 Group 1 said...

Hello Tan Siew Ming! (:

PCN is an aerobic culture for fluid extracted from the kidneys. It is similar to the urine culture.

For your second question, UTI is commonly cause by one pathogenic microorganism. It is uncommon to have more than one, though still possible. One way is to observe the appearances of colonies that grow, and also the haemolysis on blood agar. Different colonies formed could indicate a possible contamination.
Another situation could be growth is seen on CLED, yet little or no growth is seen on blood agar. This most probably results in streaking on CLED media first, then dipping back into the urine and streaking on blood agar. CLED has inhibitory properties of certain microorganisms.

Jeremy.

Hi Joanna,

MacConkey agar is a selective differential media. Due to its selective property, it might inhibit pathogenic microorganisms from growing, thus preventing diagnosis of the UTI. Thus blood agar is a more popular choice on agar plates.
For the dipslide case, the area is so small that it is inappropriate to use blood agar. Blood agar is an enriched media and will result in many colony growths. This makes it very difficult to make inferences due to its small area. MAC is preferred in this case.

Both dipslide and agar plates have their advantages and is used in different circumstances.

Jeremy.

Hi Kenneth,

Urine culture requires only the used of 1 micron wire loop. For PCN procedures, it requires both 1 and 10 micron wire loop on two separate plates. The reason for using the 10 micron loop is that sometimes, plating using 1 micron loop for PCN will not yield any colony growth.

Jeremy.

If anyone is unsure or still has any further questions, do ask. I will do my best to answer them correctly. Thank you!

TG01-Group 2 said...

Ohhh...

I get it now, thanks! :D

Siew Ming
0702862D

Ms_chew said...

Good. You are very prompt in replying the questions posted by your peers. Keep up the good work.

emadtechs said...

Hi Jeremy,

Do you proceed to identification of the organism on the agar plate straight after the overnight incubation at 35 degree celsius? Does it matter if the urine specimen is too diluted? Thanks.

Hui Juan
0702012F

TG01 Group 1 said...

Hi Hui Juan,

Identification is carried out in the investigation lab the following day or when incubation is completed.

It does not matter if urine is too diluted.

Thank you for your question.
Jeremy.

sesame chicken (: said...

Hi Jeremy,

Wanted to ask you a question :) What is the difference between using a 1 micron and 10 micron wired loop? Why direct plating require the use of both 1 and 10 micron wired loop on 2 separate split blood/CLED agar plate?

Thanks:)

Lok Pui

Dr Alex Lee said...

Hello Jeremy,

Escherichia coli is one causative agent of UTI, but does the presence of the bacteria an indication of UTI or is there a certain cfu/ml that must be obtained in order to confirm UTI?

Dr Lee

TG01 Group 1 said...

Hi Lok Pui,

Using both 1 and 10 micron wire loops only applies to the PCN method. This is because fluid from the kidneys is more sterile compared to urine from the bladder in a case of UTI. The reason for the 10 micron loop is to inoculate more specimen onto the plate to observe growth, as sometimes in PCN, plating with 1 micron loop does not yield any colonies.

Thank you.

Hello Dr Lee,

The presence of Escherichia coli could also be an indication of cystitis (inflammation of the urinary bladder), besides UTI.
In order to confirm a UTI case, cfu/ml of Escherichia coli obtained must be more than 100,000.

Thank you. Please do correct me if I am wrong.

Jeremy

Dr Alex Lee said...

Hello Jeremy,

How do you determine the cfu/ml from a patient's urine sample?
If there are 10e04 cfu/ml of E. coli, does this rule out UTI? Explain please. :)

Dr Lee

TG01 Group 1 said...

Hello Dr Lee,

Although my lab is only in-charge of culturing and not investigation of the cause of disease, this is one method of how I understand determining cfu/ml of a sample can be done:

A 10-fold dilution is created (1ml urine, 9ml water). Further serial dilutions are performed, preferably to 10e(-6). A volume of 0.1ml is used to perform direct plating and then sent for incubation. After incubation, you would expect to see colonies formed.

Plates with lower than 30 colonies are not considered significant, while the same goes to those with more than 400 colonies. The preferable range would be 50-250.
So now, you will have to count the colonies and then determine the cfu/ml by dividing the number of colonies counted, over the volume plated times dilution factor.

Formula:
No. of colonies / (volume plated) * (dilution)

An example:
Lets say we counted 80 colonies on the 10e(-5) plate.
Using the formula,
80 / (0.1ml) * (10e(-5)) = 8 * 10e(7)

This is how cfu/ml of a sample can be obtained, though it might be time consuming and tedious.

Regarding your second question, 10e04 cfu/ml does not rule out UTI, and it also does not confirm UTI. The confirmation value of cfu/ml has to be >100,000 (10e05).
This is because the value of >100,000 cfu/ml has a higher specificity of a true diagnosis compared to >10,000 (10e04). Values between 10,000 to 100,000 are seen as suspected cases of UTI.

Jeremy.

Sesame Chicken (: said...

Hi Jeremy,
are there any other combinations of agar that can be used besides the one that you mentioned? Thanks!

zi shuang :)

TG01 Group 1 said...

Hi Zishuang,

As a medical technologist, defining the cause of the disease is our main purpose. Up to date, these medias are most appropriate for detecting the causative agents of UTI. It may be possible in the future that different forms of medias may be used.

Jeremy

Sesame Chicken (: said...

ooo haha..i understand le..thanks:)

Lok Pui

MedScientists of Grp 6 said...

Hey Jeremy!

for the urine culture, if i'm not wrong, the only agar plates your lab uses is blood agar and cled is it?

becos i've been to the the urine bench in my lab, we use a combination of 3, blood, mac and cled agar.

Oh! and also, we do a quarter streak on the plate only, if yours half or quarter of the plate. (super hard to phrase it, i hope you understand what i mean :)

Janice Yeh
0701885F

TG01 Group 1 said...

Hi Janice,

There are generally 2 kinds of specimens we received commonly. Urine/PCN fluid or dipslides.
Here in my lab, we use a split blood & CLED agar for the urine sent. We do not do streaking on MAC agar.
However, for dipslides cases, MAC & CLED is used.

I do not understand the terms quarter streak or half streak. Anyway, we streak on split blood & CLED agar (half plate blood, the other half CLED) as seen in the self-drawn picture I've posted.

I hope I answer your question. If you do have anymore doubts, please do ask.

Thank you.
Jeremy