Microscopy is an interesting, straightforward and fast point-of-care method of diagnosis in Sexual Health clinics. In this article, we’ll go through the process of collecting a sample, preparing a slide and diagnostic features of different conditions.

Collecting a sample for microscopy

Samples can be taken from a range of sites; urethral, vaginal, cervical and rectal to name a few. Different swabs are used depending on where you’re sampling from, here are some examples:

Preparing a sample for Microscopy

Your next step is to prepare your sample for microscopy. This can be done as a wet mount (the sample is covered with a few drops of saline and examined directly) or by gram staining, which is explained below.

How to gram stain a sample

1. Add your sample to a slide. Take care not to contaminate the sample; the swab shouldn’t touch anything between being taken and being applied to the slide. 
2. ‘Fix’ your sample. Usually done by putting the slide on a hot plate for around a minute. This makes the sample more robust so it can withstand the processing we do in the next few steps. 
3. Staining- we use four different liquids to prepare our sample for microscopy. It’s sensible to do this over a sink as the stains can get quite messy.
   • Crystal violet- apply for 30 seconds, flooding the slide. 
   • Iodine- apply for 30 seconds over the top of the crystal violet, flooding the slide. This makes the crystal violet insoluble and it will remain in the cell walls of gram positive cells. 
   • Acetone- this works to decolourise the slide. If your sample has gram positive cells, it will remain violet, and if your sample has gram negative cells they will become colourless. 
   • Safarin (red counterstain)- apply for 1 minute, flooding the slide. This will make gram negative cells appear pink/red. Wash your slide under running water and gently blot dry, taking care not to wipe off the sample. 
4. At this point, you can put the slide back on the hot plate for a few seconds to absorb any excess water. You’ll then be ready to look under the microscope! Here are some examples of what your slide could look like after staining.

Diagnosing via Microscopy

Trichomonas vaginalis– done as a wet mount with a sample taken from the posterior fornix of the vagina.  Samples must be examined immediately as Trichomonas vaginalis is a protozoan infection and organisms will only be visible for 10-20 minutes after sample collection before they die. T.vaginalis protozoans have a distinctive appearance with their flagella moving around under the microscope.

Gonorrhoea– microscopy can be used for endocervical, rectal or urethral samples. The classic finding in gonorrhoea is gram negative intracellular diplococci (paired small dots) in polymorphonuclear leukocytes (the large cells with dark coloured blobs inside). It’s important to not that microscopy can diagnose gonorrhoea, but it can’t rule it out; if microscopy is negative, after diagnostic test (eg NAAT) still needs to be done. 

Bacterial vaginosis– done as a Gram-stained specimen. The classical findings in BV are clue cells (epithelial cells coated with small gram-negative/gram-variable rods) and reduced numbers of lactobacilli.

Vaginal candidiasis (thrush)– done as a Gram-stained specimen, which can show spores, pseudohyphae and hyphae. 

Syphilis– diagnosis is made using dark field (aka dark ground) microscopy. This creates a dark background and illuminates any cells in the sample. Dark field microscopy doesn’t require any staining and specimens are covered with a small amount of oil. Samples need to be examined within 20 minutes of collection as Treponemes lose their motility very quickly. Treponemes have a classic spiral shape and rotate in a corkscrew motion. 

I hope you’ve enjoyed this overview of the uses of Microscopy in Sexual Health! If you’re revising for medical school exams or the UKMLA, Mind the Bleep’s Finals series can help you master high yield topics. Here’s a link for a free webinar on diagnosis and management of STIs: Final Year Series: STIs and Bacterial Vaginosis UKMLA – YouTube

Written by Dr Gaayatri Sharma (F1)

With thanks to Dr Harriet Howard (ST1 CSRH) for reviewing and validating this article.