Topic: Histoplasma capsulatum
This week I will be sharing about Histoplasma capsulatum. Histoplasma capsulatum is a yeast type of fungus that primarily infects the lungs and may spread to other parts of the body. It causes an infectious disease known as Histoplasmosis. Some of the signs and symptoms are fever, headache, chest pain, dry cough and chills.
Immunodiffusion method is used to detect histoplasma capsulatum antibodies in patients’ serum. Patients’ serum and the control antigen are placed in opposite wells and diffuse outward into the medium. To a point of equivalence concentration of the antigen and antibody, two visible precipitin lines (M and H) are formed between the two wells. M line forms near the antigen well while H line forms near the serum well.
Materials and Reagents used:
1. Glycine buffered agarose agar: a diffusion medium that does not support the growth of molds.
2. Positive control: antiserum that contains H and M antibodies specific to histoplasma capsulatum.
3. Negative control: antiserum that does not contain any specific antibodies.
4. Control antigen: Contains H and M antigens from histoplasma capsulatum.
Specimen: Serum
Methods
1. Spin down the specimen at 3100rpm for 15mins.
2. Place the template underneath the Petri dish.
3. Using a hollow pin to punch the agar and a pointed stick to pick out the unwanted agar to obtain 7 wells.
4. Add about 8ml of each reagents into the respectively wells in the following order:
a. Firstly, add positive control into wells #1 and #4.
b. Negative control into well #6.
c. Patients’ serum into wells #2, #3 and #5. (Depending on the number of specimens requested for this test)
d. Lastly, add control antigen into the centre well.
[Add the reagents in this order so as to prevent any contamination that may lead to false results.]
[Prevent overflowing from one well to another as this may also lead to false results.]
5. Place the Petri dish in a moist condition at room temperature for 23-25 hours.
6. Examine the agar over a light source. (Eg. Under a microscope)
Results
Positive controls: Two lines formed between wells #1 & 4 and the centre well. [This test is valid]
Negative control: No lines formed between well #6. [This test is valid]
Patient 1 (well #2): Positive reaction as two lines are formed between well #2 and the centre well.
Patient 2 (well #3): Negative reaction as there are no lines formed between well #3 and the centre well.
Patient 3 (well #5): Positive reaction as M line is formed between well #5 and the centre well.
LeeJin
TG02
13 comments:
Hi,
I am just wondering, how do you ensure a moist condition condition at room temperature when incubating the petri dish and what is the purpose of placing it in a moist condition?
Thanks,
Xin Yi
TG02
Hi Lee Jin,
How Histoplasma capsulatum infect the lungs; is it present in the air and enters the lung when we breathe?
Is 3 the maximum number of patients'serum can be tested in one petri dish? And is it compulsory that the positions of the controls and patients' serum are exactly as the template you provide (eg. +ve control at well 1 & 4, -ve control at well 6)? If yes, why?
Thanks,
Hardina =)
Hello Lee Jin,
Wanna ask erm how does the Glycine buffered agarose agar work in such a way that it prevents growth of molds?
What does it contain? Any ingredients inside it that resist growth of molds?
Thanks,
Lloyd
hi lee jin, interesting microbio stuff =D
niwae, i would like to know the difference between concluding the patients results.
patient 1 has two lines, both H n M, while patient 3 has M line.
does this mean that patient 3 is lacking in anything? because it could only get the M line.
thank you.
raihana~
To Xin Yi
A moist condition is created by filling the container with water and place the agar plate on top of the sponge. As 3.5ml of commercially prepared glycine buffered agarose agar is added into the 5.5cm diameter petri dish (much smaller than what we normally encountered in school) the layer of agar is quite thin. In order to prevent the agar from dehydrating during incubation, a moist condition is necessary.
To Hardina
Histoplasma capsulatum grows mostly in contaminated soil, dirt and animals droppings such as pigeons and bats. H. capsulatum is a thermally dimorphic. It exists in two forms – conidia & yeasts at different temperatures (25°C and 37°C). At room temperature, it will form macroconidia and microconidia. When exposed, it may be inhaled and enters the alveolar spaces of the lungs and transforms & multiply into yeasts.
Yes, 3 specimens is the maximum. For the positions, it is not necessary. The positive controls can be placed in wells 3 and 6 or 2 and 5, as long as the two positive controls are opposite each other and all the patients’ serum are placed adjacent to the positive controls. (so either the left of the right side of the patients’ serum will be the positive control). With this arrangement, if the specimen is positive, an unbroken line of identity with the positive control can be seen.
LeeJin
Hi there...
im so sorry but i really dun get it..
how are the M and H lines formed? why M line near Ag while H line is near serum well?
thanks for sharing =)
Nur Farhana
0604834B
hi lee jin, assuming that the hospital needs the results urgently, is it possible to shorten the incubation time of 23-25 hours? Perhaps by increasing the temperature or other factors?
cornelyus
To Raihana
The clinically significant antigens of Histoplasma capsulatum are M and H antigens. Each visible precipitin line represents different antigen. The line closer to the centre of the well is known to represent M antigen whereas the other line represent H antigen. One or two precipitin lines can be seen.
Patient 1 has two lines – H and M. This means that there is a present of both H and M antibodies in the patient’s serum. It commonly occurs during active histoplasmosis.
Patient 3 has 1 line – M, means that only M antibodies are present in the serum. This may be due to active infection or post infection.
H line can be present alone but this is quite rare and its clinical significance is still uncertain therefore it is not mentioned.
To Lloyd
Histoplasma capsulatum is a dimorphic organism. It can be yeast or mold at different temperatures. At room temperature, it is a mold. In body temperature, it transforms to yeast. As the plate incubates at room temperature, it is more likely to become mold and also molds grow best in acidic condition. Glycine buffered is the buffering agent used to prevent mold from forming by changing the pH to alkaline condition thereby preventing mold from growing.
LeeJin
To Farhana
This test uses immunodiffusion method. The principle of this method is similar to the method under precipitin reactions taught in AIMM.
M and H lines are formed due to antibody – antigen reactions whereby the control antigen and patients’ serum diffuse from the wells. If the patients’ serums contain the antibodies specific to the control antigen, it will reach to a point of equivalence and a visible line of precipitin forms. If there is one line formed, this means that only type of antibody is present in the patients’ serum. 2 lines formed means 2 types of antibodies present.
M antibody is considered the more significant one. It is mostly the first to be detected so there will be a higher concentration as compared to H antigen and M line appears closer to the control antigen well. Therefore the H line will form farther away from the antigen well.
To Cornelyus
It is not possible to shorten the incubation time. Time is needed to allow the antigen and antibody to diffuse through the medium and give a reaction. In Fungus lab, most of the tests need at least two weeks of incubation. This test is considered one of the fastest.
LeeJin
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