Procedure: Biochemical Testing for Neisseria gonorrhoeae 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
And plain blood specimens.
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
Only the first three are confirmatory tests. The last test is a susceptibility test to check for 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)
Rapid Carbohydrate Degradation Test (Sugar Test)
Principle
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.
Procedures
- 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
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!!!