Taylor-Leigh Siebritz
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Microscopy
Microscopy
The use of microscopic imaging is the most common and simplest form of diagnosis for parasitic, bacterial and even some virologic agents of disease. Giardia trophozoites or cysts in stool specimens are identified using different types of microscopy.
Figure 20: Unstained G. lamblia cyst, viewed using Bright-Field Microscope (BFM) at 40x magnification (DPDx, 2020).
For Giardia lamblia, microscopy is performed by direct smears, which excludes the need for concentration techniques,and uses dry- or wet mount preparations instead. However, these can be necessary when low parasitic numbers are present in the sample (Alharbi, et al., 2020).
Dry mount preparation:
Dry mount preparation:
This simple slide preparation involves only mounting a clean coverslip over a thin smear of the stool sample on a sterile microscopy slide (Vicente, et al., 2024).
Wet mount preparation:
Wet mount preparation:
Saline wet mount:
On a sterile microscopic slide, a small drop-full of 0.85% saline is placed and mixed well with a tiny amount of the stool sample. On top of this, a clean coverslip is placed. Light microscopy of the specimen at 10x and 40x magnification reveals the presence of parasites. Trophozoites are typically found in diarrheic stool, in saline wet mounts (Kumar, et al., 2022).
Iodine wet mounts are prepared and observed similarly, with the use of Lugol's Iodine in place of simple saline. Cysts are usually found in formed stool, and are visualised well with Iodine wet mounts (Kumar, et al., 2022).
Modified Ziehl-Neelson (ZN) Staining:
Air-dried thin stool smears are fixed with methanol for 3 minutes before staining with and heating with concentrated Carbol fuschin until steaming. A rinse with tap water and 1% acid-alcohol decolourisation for 1 minute is then performed, followed by a counterstain of Methylene blue for another minute. Viewing under light microscopy requires 100x magnification and immersion oil (Kumar, et al., 2022).
Concentration Techniques
Concentration Techniques
The detection of Giardia lamblia parasitic forms by sample concentration includes two methods (Alharbi, et al., 2020):
Ritchie (Formal Ether) Sedimentation Technique:
An emulsified solution is produced from the sample by mixing 2mg of sampled stool with 10ml of 10%-formal-ether. Filtration and centrifugation at 2000rpm for 5 minutes is then performed to produce a supernatant and sediment. The supernatant is discarded, while the sediment is re-suspended in another 10ml of 10%-formal-ether solution, and centrifuged at the same settings again with an added 3ml of diethyl-ether. This produces a four-layered substrate, of which the top three are discarded. The remaining layer, the sediment, is then mixed with a drop of Lugol's Iodine. Viewing under Light Microscopy requires 10x or 40x magnification.
Para-Pak Trichome Staining Technique:
Fixation of the with Polyvinyl Alcohol (PVA) precedes the addition of Trichome stain. After 6-8 minutes of air-drying, the smears are immersed in acid-alcohol for 5 minutes, then in (95%) ethanol for another 5 minutes, and in (96-100%) ethanol for 3 minutes. Finally, the slide is immersed in xylene for 3 minutes, before a coverslip is mounted. Viewing under Light Microscopy requires 100x magnification with immersion oil.
Figure 21: Bright-Field Microscopy (BFM) of G. lamblia trophozoite (DPDx, 2020).
Figure 22: Bright-Field Microscopy (BFM) of G. lamblia cyst (DPDx, 2020).
Figure 23: Iodine-stained G. lamblia cyst, viewed under Bright-Field Microscopy (BFM), at 40x magnification (DPDx, 2020).
Advantages of Microscopic Detection: (Fitri, et al., 2022)
Advantages of Microscopic Detection: (Fitri, et al., 2022)
simultaneous detection of multiple intestinal parasites
affordable, and long-term cost-effectiveness
ease of implementation, and easily taught skills
Disadvantages of Microscopic Detection: (Fitri, et al., 2022)
Disadvantages of Microscopic Detection: (Fitri, et al., 2022)
low sensitivity due to the intermittent excretion of cysts in feces
multiple sample examinations required for enhanced parasitological diagnosis
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