Posted by: Azhar Hamdan (0503269C)
For the moment, I am temporarily attached to the Serology Lab for 2 weeks, with my first week being in the STD Lab. Yeap, thats rite Sexually Transmitted Diseases. I know what you are thinking, "Eeeks HIV!" However, on the contrary , the lab only tests for 2 organisms, Neisseria gonorrhoeae (causes Gonorrhoeae) and Treponema Pallidum (Syphilis). So no HIV here :).
For this post, I am going to try adding as much pictures as possible to keep you guys interested alrite?
Anyways my post will focus more on N. gonorrhoeae, a.k.a Gonococci or GC.
The common types of specimens received by the lab are swabs from:
- Endocervix
- Urethra
- Anorectal
- Oropharynx
- Conjunctiva
- Bartholin gland
- Skin lesions
- Joint fluids
An interesting point to note is that more than half of the specimens come from Sex Workers that regularly go for checkups to retain their license. So people, always stay protected!
Anyways, upon receiving the specimens, the lab staff would process the specimens and allocate lab numbers. They would then inoculate & streak the swabs onto GC -Lect Agar, a selective agar that promotes the growth of GC that contains antibiotics that would suppress the growth of other organisms such as gram-positive bacteria, including vancomycin-resistant Staphylococcus epidermidis, gram-negative species like Proteus and Capnocytophaga, as well as fungi including Candida albicans. The plates would then be incubated at 35-37°C for 24 hours.
All the above is done by the lab staff.
After incubation, the plates are read. Suspected GC colonies undergo a Gram-stain & 4 biochemical tests (these were practised by us students):
- Oxidase test to test for the production of oxidase
- Superoxol test (using 30% Hydrogen Peroxide [H2O2]), similar to the Catalase test (using 3% H2O2), to test for the production of catalase
- Rapid Carbohydrate Degradation Test (Sugar Test), to test for the ability to degrade sugars)
- β-Lactamase test, to test for the production of penicillinase which confers resistance to penicillin
The oxidase test and superoxol test (similar to catalase) are some common tests that we have learnt in Basic Microbiology so I shall not go into details, however, questions are welcomed.
I chose the Rapid Carbohydrate Degradation Test (Sugar Test) to elaborate on, as it is so different from all the other tests that checks for the utilisation of sugar (e.g. TSI & KIA). Specimens are tested with the sugars individually. The sugars used in this test are 10% solutions of Glucose (G), Maltose (M), Lactose (L) and Sucrose (S)
This is a non-growth method which depends upon pre-formed enzymes. A heavy inoculum of organism degrades the sugar solutions containing the phenol red indicator. A colour change occurs in 2 to 4 hours.
- A heavy suspension of the organism is made (using a platinum inoculating loop) in a test tube of 1.0mL Buffered Salt Solution (BSS), to contain approximately 109 organisms per mL. Controls were also done together with the patient specimen. These controls are N. gonorrhoeae CDC 117 strain (117), N. lactamica (LC), Branhamella catarrhalis (BC) & Staphylococcus epidermidis (S)The BSS is red in colour due to the phenol red indicator.
- This suspension is mixed well with a Pasteur pipette (Two Pasteur pipettes are available, use the bigger pipette [top] for this step).
- Using the bigger Pasteur pipette, two drops of the suspension are delivered into each of the five wells of a microtitre plate which are labeled horizontally across the plate as Control (C), Glucose (G), Maltose (M), Lactose (L) & Sucrose (S).[The group of wells on the left side were from a previous practice, microtitre plates are shared to avoid wastage]
- Using the smaller Pasteur pipette, one drop of each of the 10% carbohydrate solutions (G, M, L and S) is added carefully to each of the appropriate wells, making sure there is no splashing.
- Cover the microtitre plate and mix well using the micro-shaker.
- Incubate at 35-37°C for 2-4hrs
- After incubation, examine plate for colour change. A red to yellow colour change indicates a positive reaction. Control well should remain an orange/red colour which indicates a negative reaction.
Results for the above test (both left & right specimens):- First row, patient specimen: positive for G.(N. gonorrhoeae)
- Second row, 117: positive for G.
- Third row, LC: positive for G, M & L.
- Fourth row, S: positive for G,M & S
- Fifth row, BC: all negative
- First row, patient specimen: positive for G.(N. gonorrhoeae)
All the results tally with the expected results.
Pictures were taken with permission from the supervisor. Thanks Andre for being my model. Haha.
So thats all.
5 more weeks...Ahhhh!!!
12 comments:
Azhar!!
That is so intersting.. Just want to ask why is a platinum inoculating loop used and how do you determine that there are 10^9 organisms per mL? And also, all these can be done on an open bench, no need to be done inside a BSC?
Thanks=]
Ming Boon
0503197F
Tg01
Hi Azar,
Do all the samples/swabs undergo tests like oxidase tests, superoxol tests etc or different types of samples obtained will be pre-designated for different type of tests?
Out of curiosity, does your lab perform TPHA( syphilis testing) using automation? Because the infectious disease lab that I'm attached to performs HIV, Hepatitis B & C and TPHA testing using machines.
From the pictures you've taken, it seems that experimental work are not done in a lamina flowhood. What preventive measures do you all take when handling potential hazardous samples besides the basic wearing gowns, gloves and goggles etc?
I enjoyed reading your post. Cheers! =)
Loh Sharon, Tg 01
Hi
You said that Superoxol test is similar to the Catalase test, but what are the differences between them? Thanks
Eugene Wong
TG02
Hi Azhar
That was so cool! I wanna know are the 3 confirmatory tests specific to the detection of GC colonies? and what's the pupose of the buffered salt soltuion? Thanks!
June, TG02
Hey azhar,
juz wondering if N. gonorrhoeae can penetrate through skin? if so then u muz be careful..ok anyway my ques, y is there need to test for maltose? frm MMIC we oni do sucrose, glucose and lactose rite..
Tanx!
Nisha
TG02
0503254E
hey azhar...
u mention that the results will be out in 2 hours. however, for normal sugar degradation test it requires 4 days of observation to confirm the results. just wondering why is it like that?
~Jeremy~
TG01
Hi Azhar!
May i ask what and where is Bartholin gland found?
Thanks!
Yvonne
tgo1
hihi
that is interesting, you mention about the GC -Lect Agar, a selective agar that promotes the growth of GC that contains antibiotics that would suppress the growth of other organisms. What the antibiotics that this agar contains?
Juexiu
tg02
hey azhar,
how do u conduct the β-Lactamase test?
thanks
andre
tg01
hey azhar,
u mention that the suspension should contain approximately 10^9 organisms per mL. but what if the organism (staph) is capable of producing high amounts of enzyme that can break down the sugar. do you still inoculate so much?
boonching
Answers:
To Ming Boon:
The use of a platinum loop is not essential for the sugar test, it does however affect the oxidase test. A nickel or wire loop that contain iron may cause a false positive oxidase test. Since all of these tests are done back to back, it is just easier to just use one loop.
One way to determine whether there are 10^9 organisms per mL is to use a turbidity standard such as the McFarland Equivalence Turbidity Standard to compare the turbidity of your suspension. However, the estimated amount of colony to be picked up is about half a loop.
To Sharon:
All the samples/swabs are plated upon receipt and then incubated. After incubation, the plates are read and only suspected colonies undergo ALL the tests. Some related species such as N. lactamica may produce a similar oxidase test to N. gonorrhoeae, but it is weakly positive for the superoxol test unlike N. gonorrhoeae which is strongly positive, thus all the tests have to be performed to confirm the identity of N. gonorrhoeae.
As for TPHA, this lab does perform that test but it is not automated. Our test is performed manually and is a variation of TPHA. It is instead called TPPA(terponema pallidum particle agglutination) as it employs particle agglutination.
Besides the basic PPE such as labcoats and gloves, masks are used. Other than that, basic safety precautions apply, such as not touching any part of your body with your gloves. Don't mind some of the pictures where no masks were worn, those are for demonstration purposes only. The organism was not handled during those periods.
To Eugene:
The main difference between catalase and superoxol test is the concentration of Hydrogen peroxide(H2o2) used. For Superoxol, 30% H2O2 is used whereas for Catalase test, 3% H2O2 is used.
To June:
These 3 confirmatory tests can be said to be specific for the detection of GC colonies, however further tests are performed to confirm that the colonies are GC. Examples include gram staining and employing the Immunofluorescence technique using antibodies specific to GC. The purpose of the buffered salt solution is mainly as a media for creating the suspension of GC. However, the buffered salt solution also contains the phenol red indicator which changes colour when the sugar is degraded.
To Nisha:
N. gonorrhoeae cannot pass through the skin just like other bacteria. However, it can infect the eye and it can also enter your blood through open wounds, thus it is always important to wear PPE when handling N. gonorrhoeae.
As for your next question, sugar degradation/fermentation is not just limited to the three sugars you mentioned (sucrose glucose and lactose). If these 3 sugars are absent in the environment wherease maltose is abundant, it would make sense that some organisms would be able to degrade/ferment the maltose to survive in those conditions. From the last picture, you can see that the related strain N. lactamica is able to degrade glucose maltose and lactose, wherease N. gonorrhoeae only degrades glucose. Thus it is important not only to detect that glucose can be degraded, t is also important to be able to detect that the other sugars, maltose, sucrose and lactose are not degraded.
To Jeremy:
As mentioned in my post, this test 'is a non-growth method which depends upon pre-formed enzymes'. Since it is a non-growth method, it does not need to be incubated and observed for a few days. Usually those few days of observation are required just in case the organisms have a slow growth.
To Yvonne:
Well, the Bartholin's glands, as i myself just found out, are glands located near the opening of the vagina. So these specimens are thus received from females only.
To Juexiu:
Well im sorry to tell you this, but the identity of the antibiotics present in the agar are not revealed by the manufacturer. All that is known is that there is vancomycin present.
To Andre:
The test goes like this:
A piece of filter paper soaked in bromocresol purple penicillin solution (BCP) is placed in a petri dish. Using a bacteriological loop, touch a few colonies of the organism and spread it over an area approximately 5mm in diameter on the filter paper. Colonies producing penicillinase produce a yellow zone, while those that do not produce penicillanase do not give any yellow colouration. A positive test as indicated by colour change, is usually visible 1-10min but the test should be examined for at least 30min.
To Boon Ching:
If the organism is capable of producing high amounts of enzymes, lesser organisms were used to make the suspension
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