Hi guys I’m attached to Microbiology department for 20 weeks. This department consists of various labs. So for the first 5 weeks, I will be in Central Processing Lab. This lab processes the specimens upon receiving it. Wanna share with you how we process urine and stool specimens.
Urine Culture
Principles:
- Urine culture is done to detect bacteriuria.
- Urine is cultured onto the media by surface streaking. It will then be incubated to promote the bacterial growth and obtain single colonies and allow viable count.
- It helps in the diagnosis of Urinary Tract Infection (UTI).
- Primary cause of UTI: members of Enterobacteriaceae family. Some possible causing agents are Escherichia coli, Proteus spp. and Klebsiella spp.
- Fresh midstream urine should be used.
- Specimens come in a form of urine bottle or Dip slide container.
Materials:
1. Patients’ urine specimens
2. BAP & CLED plates
3. Dip slides
4. Disposable inoculating loops (1µl and 10µl)
Picture taken from http://canadawide.ca/products/596-300.1.html
5. Disinfectant container , Biohazard waste bin
Procedures:
For specimens in urine bottles:
1. Dip a 1µl disposable inoculating loop vertically into the specimen.
(If the urine specimens come from nephrostomy or collected in the Operating Theatre, an addition of 10µl disposable inoculating loop should be used. So a total of 2 BAP and 2 CLED agar plates are used.)
2. Perform surface streaking onto BAP and CLED agar plates.
Surface streaking
1. Streak a vertical line down the agar plate.
2. Streak closely from the top. Make wider streaks as you move downwards to obtain single colonies.
1. Streak a vertical line down the agar plate.
2. Streak closely from the top. Make wider streaks as you move downwards to obtain single colonies.
3. Discard the disposable inoculating loops into a disinfectant container.
4. Discard the specimens into the biohazard waste bin.
5. Incubate all the agar plates at 35°C overnight.
For specimens in dip slides:
1. Incubate it at 35°C overnight after receiving the specimens.
After incubation, all agar plates and dip slides are sent to the Investigation Lab to quantify and identify the colonies if there is any bacterial growth.
Stool Culture
Principles:
- Stool culture is done to identify the cause of infection in the intestine, diarrhea or bloody stool.
- It is commonly used to identify Salmonella, Shigella, Campylobacter and Vibrio spp.
- Diarrhea is usually caused by Vibrio spp. so an addition of Thiosulphate-citrate bile sucrose agar (TCBS) and Alkaline Peptone Water (APW) enrichment broth are used to enhance the growth of this genus.
- Selenite F broth is an enrichment broth for the growth of Salmonella spp.
- Bloody stool is caused by enterohemorrhagic Escherichia coli (EHEC). Commonly by E. coli O157:H7 strain. Sorbitol MAC media is used to differentiate E. coli O157:H7 from other E. coli strain.
Materials | |||
1. | Patients’ stool specimens | ||
2. | Inoculating loops | ||
For solid stool | For watery stool | For bloody stool | |
3. | Blood Agar Plate ( MacConkey agar ( Salmonella–Shigella agar (SS) Campylobacter agar Selenite F broth | ||
4. | |||
5. | |||
6. | |||
7. | |||
8. | TCBS | Sorbitol | |
9. | |||
10. | Wooden stick | Cotton swabs | |
Procedures:
1. Use wooden stick / cotton swab to take up the specimen and streak onto the appropriate agar plates. Then place it into Selenite F broth and/or APW.
1. Use wooden stick / cotton swab to take up the specimen and streak onto the appropriate agar plates. Then place it into Selenite F broth and/or APW.
2. Use inoculating loops to perform tertiary streak for all agar plates.
For Sorbitol MAC agar, a control is done to ensure that the agar is functioning.)
3. Discard the inoculating loops into a disinfectant container.
4. Incubate all media at appropriate conditions.
4. Incubate all media at appropriate conditions.
Media | Incubation |
35°C , 18 – 24 hours | |
SS | |
TCBS | |
Selenite F broth | 35°C , 12 – 18 hours |
35°C , 6 – 12 hours | |
Sorbitol | 35°C , 24 hours |
Campylobacter agar | 42°C , 48 hours with 85% N2, 10% CO2 and 5% O2 |
5. After incubation, Selenite F broth and APW are sub cultured and then incubate it.
6. After incubation, all the plates are to the Investigation Lab to identify the colonies if there is any bacterial growth.
Posted By:
LeeJin
TG02
14 comments:
hey leejin!
Can i ask why must we perform urine culture on BAP and CLED agar plates?
thanks!
cheers,
huimin =)
tg01
Hi Huimin
BAP is a general purpose media that allows the growth of most of the microorganisms that causes UTI. It also allows the growth of fastidious organisms. If Proteus spp. is present in the urine, it will grow and swarm the surface of BAP which makes it difficult to differentiate and enumerate the colonies.
CLED agar plates which lack of sodium chloride will help to reduce Proteus swarming. It is commonly used in the isolation and differentiation of urinary tract bacteria. So it is useful to use BAP and CLED agar plates.
LeeJin
TG02
Hi LeeJin,
1)Why should midstream urine be used?
2)"(If the urine specimens come from nephrostomy or collected in the Operating Theatre, an addition of 10µl disposable inoculating loop should be used. So a total of 2 BAP and 2 CLED agar plates are used.)"
Why is there a need for an additional 10µl disposable inoculating loop to be used if the urine sample come from nephrostomy or collected in the Operating Theatre?
3)Why is surface streak preferred over normal agar streak which we normally performed in BMic/MMic?
Thankz :)
Han Yang
TG01
Hi LeeJin
Hope it's somewhat a sunny day over at your attachment place =)
Just 2 quick questions:
1) You mentioned that CLED agar plates reduce Proteus spp. spreading. How does that helps in better differentiation and identification of the bacteria?
2) You mentioned that for Sorbitol MAC agar, a control is done to ensure that the agar is functioning. Is that a positive control or negative control?
Thanks a million!
Quan Jun
TG02
Group 08
Posted: 14 July 2008
Hey Lee jin,
are u enjoying ur SIP so far? Hope that u are =)
Anyway, i am also attached to the micro lab for the 1st month of my attachment, haha.. so the test that u mentioned is similar to the test i do in the lab i am attached to.
Just curious, for the urine samples u mentioned that is to be steak to the blood agar, do u also steak on the MacConkey agar? Cos in my lab we do that for every urine samples received.
Oh ya, do u only do control for the Sorbitol MAC agar? How come? Is it because there is high chance of the agar not functioning properly or just purely to have more accurate results?
Tks =)
Have Fun.
Hi Lee Jin! Are there any other types of organisms or causes of UTI apart from those from the Enterobacteriaceae family?
Any examples?
Enjoy SIP. (:
Elyana
0606676E
TG01
Hi Han Yang
1. Due to the flow of the urine, the starting and the ending of the urine will have a higher chance of being contaminated so it is better to use midstream urine.
2. Nephrostomy is a surgical procedure that drains urine from the kidney to a nephrostomy tube. Urine obtained from these areas is considered precious as it is difficult to obtain it again. So an addition of BAP and CLED plates for 10ul disposable inoculating loops are used so as not to miss any microorganisms present in urine.
3. Urine culture is done mainly to quantitate the colonies so as to identify which microorganisms in the urine are associated with the infection. This method of streaking allows the colonies to be counted easily than the usual method. The usual method is more to isolating of colonies whereas this method mentioned is more to counting of colonies.
LeeJin
TG02
Hi Quan Jun
1. The growth of Proteus spp. will swarm the medium surface of BAP. If other microorganisms are also present, Proteus spp. will cover the other colonies. The colonies cannot be picked up and thus it is difficult to identify and differentiate the type of microorganisms. CLED (Cystine lactose electrolyte deficient) plates which lack of sodium chloride will inhibit Proteus spp. swarming on the medium surface thus allowing other colonies to be seen clearly and so it can be picked up easily for identification.
2. The control used is E. coli O157:H7. As Sorbitol MAC agar is used to detect E. coli O157:H7 therefore it is a positive control.
LeeJin
TG02
Hi Zhenling
For us, we only streak onto BAP and CLED. BAP is a general purpose medium that grows most of the microorganisms that cause UTI so BAP is good enough to detect the possible microorganisms so we don’t streak onto MacConkey agar.
For stool cultures, we do control for Sorbitol MAC agar and daily quality control for Campylobacter agar. Yes mainly to have more accurate results and also to ensure that the incubation condition is right. This will confirm that the patient is infected with E. coli O157:H7 strain if there is growth in both the control and the patient’s specimen.
LeeJin
TG02
Hi Elyana
There are other types of organisms that cause UTI too. Two examples are Pseudomonas aeruginosa and Candida albicans. But the most common ones are the organisms from Enterobacteriaceae family.
LeeJin
TG02
Lee Jin. Very good. You have answered the questions posed promptly.
Hello Lee Jin
Hi, hope you have been enjoying your SIP very much? Btw i have some questions to ask you ...
1) How will colonies appear on BAP and CLED after culture from the urine bottle?
2) Why are disposable inoculating loops used instead of the metal one?
3) Do you need to decontaminate/sterilize all biological wastes? If so can you list the autoclave/decon procedures?
4) How can Sorbitos MAC differentiate O157:H7 E coli from other strains of E.coli
5) Why is there a need to perform subculture for Selenite F broth?
Thanks for answering my questions :)
From: Ma Xianwei Benjamin
Class: TG01
0606181F
Hi Benjamin
1. After incubation, all the plates will be sent to the investigation lab to check for any pathogenic bacteria. I have yet to see the plates as I will only be going to the lab next week. Meanwhile I will try to get the information if not I will let you by next week. Sorry for the delay.
2. Using disposable inoculating loops are more practical as there is no need to heat the loops and there is no need to wait for the loops to cool down before proceeding to the next specimen. Each day there are a lot of specimens needed to process so using the disposable ones will save a lot of time. The specimens are processed in the biosafety cabinet and the loops are placed inside the BSC before opening the package. It is considered sterile so contamination will not be an issue.
3. For urine and stool specimens, there is no need to decontaminate or sterilize them. A license contractor will collect the biological wastes and incinerate it into ashes. It will be done elsewhere so I am not sure of the procedures. Sorry about it.
4. E. coli O157:H7 and other E. coli strains ferment lactose on MacConkey agar and appear as red or pink colonies. Therefore E. coli O157:H7 cannot be distinguished from other lactose-fermenting E. coli in MacConkey agar.
Sorbitol MAC agar is a differential medium that contains sorbitol instead of lactose. E. coli 0157:H7 strain is unable to ferment sorbitol thereby producing colourless colonies. The other E. coli strains are able to do so which results in red or pink colonies. So Sorbitol MAC agar can differentiate E. coli O157:H7 strain from the others.
5. Sometimes bacteria grow on the plates can be insufficient to identify. It is better to culture into the broth to enhance the growth of the bacteria and then sub cultured onto the plates to see whether the particular bacterial is responsible for the infection.
LeeJin
TG02
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