Ryane E. Englar and Jeremy Bessett Fecal flotation is a routine diagnostic test that screens concentrated fecal samples for evidence of parasitic infection. This test can recover cysts, oocysts, and eggs of mature parasites that live inside the body and reproduce [1, 2]. To perform this test, the diagnostician bathes macerated feces in hypertonic solutions of concentrated sugar or salts [1, 2]. These solutions force parasitic components, which are less dense, to float to the top, where they can be retrieved for microscopic examination [1–5]. This requires an understanding of specific gravity. Specific gravity was first introduced in Chapter 13 with regard to urinalysis. Recall that when we measure specific gravity, we are making an assessment about density [6]. Density is a measure of the mass of an object relative to the space that it occupies [6]. When we consider density of a solution, then what we are really investigating is the mass of solute per volume of solution. Fresh water is said to have a specific gravity of 1.000 at 4°C at sea level [6]. Tap water is said to have a specific gravity that is just above 1.000 [1]. Solutions that have a specific gravity less than 1.000 are less dense than water and will float. These include gasoline, automotive oil, kerosene, jet fuel, lard oil, and corn oil (see Figure 18.1). Objects that are less dense than water will also float. Consider, for instance, ice cubes, and corks (see Figure 18.2). Solutions that have a specific gravity greater than 1.000 are denser than water and will sink. These include whole milk, 5% sodium chloride, and propylene glycol. Objects that are denser than water will also sink. Consider, for instance, parasite eggs. Regardless of which parasite they come from, ova have a specific gravity that exceeds 1.000 [1]. This means that if we tried to separate ova from tap water through centrifugation in an attempt to get them to rise to the surface, we would be unsuccessful. The eggs would be too heavy. They would sink to the bottom instead [1]. The specific gravity of most parasite ova falls between 1.05 and 1.203 [1, 7]: To float ova, we must suspend them in a solution that exceeds their specific gravity [1, 2, 5, 7]. We achieve this by creating hypertonic flotation solutions that heavily concentrate sugar or salts [2]. Hypertonicity of flotation solutions is essential. However, there is such a thing as too much. If the flotation solution’s specific gravity is too high, then we run the risk of rupturing ova or protozoal cysts, or at the very least, distorting them as they lose water via osmosis to the solution in which they are being bathed [5, 7]. We also want to discourage larger particulate matter that is suspended within fecal samples from also rising to the top when placed in solution [1, 5]. This would make it challenging to discern parasites from fecal debris. We must therefore keep in mind the specific gravity of feces when selecting an ideal flotation solution. Fecal matter has a specific gravity that exceeds 1.3 [1]. Therefore, our ideal fecal flotation solution should have a specific gravity that exceeds 1.2 but is less than 1.3. This provides an efficient means by which to separate out most parasitic life stages from fecal matter [1]. Because feces are heavier than the flotation solution, they will sink along with any particulate matter contained within [1, 4]. Because eggs, larvae, and cysts are less dense than most flotation solutions, they will rise to the top for sampling by the diagnostician [1, 4]. This is achieved by positioning a glass microscope slide over top of the solution. Anything that rises to the surface of the solution will adhere to the slide. The slide can then be examined under a microscope to facilitate identification of parasites. Figure 18.1 Oil mixed with water. Note that the oil is less dense than water, so the oil floats to the top, supported by the water beneath. Source: Courtesy of Ryane E. Englar, DVM, DABVP (Canine and Feline Practice). Figure 18.2 Ice added to a cup of water. The ice is less dense than water, so the ice floats to the top, supported by the water beneath. Source: Courtesy of Ryane E. Englar, DVM, DABVP (Canine and Feline Practice). Common fecal flotation solutions that are used in veterinary practice include [1, 2, 5, 7–10]: Figure 18.3 One commercially available brand of Sheather’s sugar solution. Source: Courtesy of Ryane E. Englar, DVM, DABVP (Canine and Feline Practice). Figure 18.4 One commercially available brand of zinc sulfate solution. Source: Courtesy of Ryane E. Englar, DVM, DABVP (Canine and Feline Practice). Figure 18.5 One commercially available brand of sodium nitrate solution. Source: Courtesy of Ryane E. Englar, DVM, DABVP (Canine and Feline Practice). Specific gravity of flotation solutions should be confirmed at least monthly via hydrometers [2, 7]. This is particularly important if the solution is being prepared in‐house. Evaporation of the stock solution will unintentionally raise the solution’s specific gravity [7]. This may be undesirable, particularly if the evaporating solution’s specific gravity becomes high enough to cause rupture of cysts or ova [7]. Although the direct smear technique that was outlined in the preceding chapter (see Chapter 17) is a valuable diagnostic tool, its small sample size significantly reduces the likelihood that parasite eggs, larvae, or protozoal cysts will be identified [1]. Alternate methods that concentrate fecal matter provide additional value by increasing the chance that one or more developmental stages of parasites will be observed [1]. Fecal‐borne parasites are prevalent among companion animals. Given the zoonotic potential of many of these parasites, fecal egg shedding represents a significant public health issue [8, 12]. Zoonotic species include, but are not limited to, Toxocara canis, Toxocara cati, Ancylostoma caninum, Giardia spp., Cryptosporidium parvum, and Toxoplasma gondii [8]. Although many people associate parasitic infections with tropical regions of the world, Americans are also at risk of contracting parasitic disease [13]. In 2020, the Center for Disease Control (CDC) named five parasitic infections as priorities for increasing public awareness [13]. Based upon the sheer numbers of those infected and the severity of disease, the CDC prioritized Chagas disease, neurocysticercosis, toxocariasis, toxoplasmosis, and trichomoniasis [13]. Human exposure to toxocariasis is climbing. The CDC reported exposure of 14% of the US population in 2020 with an estimated 70 people annually becoming blind because of infection [13]. A 2016 study by Lucio‐Forster evaluated a cumulative data set from 2011 to 2014 that was compiled by the Companion Animal Parasite Council (CAPC). The goal of the study was to determine prevalence of Toxocara egg shedding from more than 500,000 feline and 2.5 million canine fecal samples [14]. Shedding of Toxocara by companion animals ranged from 0% to 18.2% in cats and from 0% to 5.3% in dogs depending upon the state of residence [14]. However, most states demonstrated a prevalence between 1% and 8% in cats and between 1% and 3% in dogs [14]. What this means is that for every 20 cats across the nation, 1 is shedding Toxocara eggs, and one out of every 60 dogs is doing the same, except for in the southwestern United States, where prevalence is higher among canine patients [14]. What is most concerning about this is that fecal samples from cats are examined less often than samples from dogs [14]. From 2011 to 2014, nearly five times as many fecal tests were submitted for canine patients as compared with feline patients, yet cats are a major source of environmental contamination when it comes to shedding of ova, particularly roundworms [14]. Lucio‐Forster and Bowman examined fecal samples from 1,322 cats from shelters and affiliated foster homes in upstate New York over a 3.5‐year period [15]. Eighteen different parasites were identified by the research team, and at least one parasite was detected in 50.9% of all samples [15]. The feline roundworm, Toxocara cati, and Cystoisospora spp. were most prevalent [15]. Twenty‐one percent of all samples contained Toxocara cati [15]. Twenty‐one percent of all samples contained Cystoisospora spp. [15]. Additional parasites and their associated percentages include: Giardia spp. (8.9%), the lungworm, Aelurostrongylus abstrusus (6.2%), ova from Taenia spp. (3.9%), Cryptosporidium spp. (3.8%), Aonchotheca spp. (3.7%), Eucoleus spp. (2.3%), Ancylostoma spp. (2.2%), Cheyletiella spp. (2.0%), Dipylidium caninum (1.1%), Otodectes spp., Toxoplasma‐like oocysts and Sarcocystis spp. (0.8% each), Demodex and Spirometra spp. (0.4% each), and Alaria spp. and Felicola subrostratus (0.2% each) [15]. Regional differences in parasite distribution have been reported. For instance, a 2019 report by Hoggard et al. documented the prevalence of parasites from shelter cats in northeastern Georgia. Flotation of 103 samples using a sugar solution disclosed eggs of Toxocara cati (17.5%), oocysts of Cystoisospora felis (16.5%), Ancylostoma sp. (11.7%), oocysts of Cystoisospora rivolta (8.7%), ova from Taenia spp. (3.9%), Spirometra mansonoides (2.9%), Mesocestoides sp. (1%), Dipylidium caninum (1%), and Eucoleus aerophilus (1%) [16]. Over one‐third of cats euthanized by animal control agencies in Northwestern Georgia (39.6%) tested positive for gastrointestinal helminths [17]. Coinfection with two helminths was present in 6.1% of the sampled population [17]. Coinfection with three or more helminths was identified in 1.1% of the sampled population [17]. These are just a handful of studies in the growing veterinary medical literature concerning gastrointestinal parasitism. These and others demonstrate that risk of feline patients acquiring intestinal parasites is real despite years of being overlooked by pet owners and veterinarians alike. For the benefit and well‐being of our patients and their caretakers, it is essential for veterinary professionals to emphasize the importance of fecal screens within the context of preventative care. We need to accurately diagnose affected patients irrespective of whether they are clinical for disease so that we can effectively implement parasite control strategies. We also need to catch and curb asymptomatic patients that contribute to environmental contamination through fecal shedding of ova. Fecal flotation is a critical diagnostic test that identifies most gastrointestinal parasites, including roundworm, hookworm, and whipworm ova as well as protozoal oocysts, such as Coccidia and Toxoplasma [4]. In addition, fecal flotation can recover digested skin mites like Demodex spp. or Cheyletiella spp., thereby contributing to dermatologic diagnosis [4]. According to the 2020 guidelines from the CAPC, fecal analysis should take place at least four times during the first year of life for puppies and kittens [18]. Thereafter, canine and feline patients should be subject to fecal examinations at least twice per year throughout adulthood [18]. Recognize that patient health and lifestyle as well as parasite prevalence within the geographical residence of the patient may further influence frequency of fecal examination [18]. In addition to routine screening, fecal analysis must be considered an essential part of the diagnostic workup for those patients that present with aberrant defecation histories and/or clinical presentations, including, but not limited to [19]: There is more than one way to perform fecal flotation. In veterinary practice, two methodologies are commonly employed [4]: (see Figures 18.6 and 18.7). Passive flotation is predominant among veterinary clinics because it is efficient, expedient, and economical: disposable kits are available for purchase commercially and you do not require a centrifuge [4]. All you need is a sample, a disposable kit, flotation solution, and a microscope. Greater detail concerning equipment and procedural steps will be provided below. Centrifugal flotation relies upon a centrifuge to separate particulate material based upon differential densities so that less dense ova or cysts float to the surface of the solution [22]. There are two types of centrifuges that you might choose to support fecal analysis in private practice: (see Figures 18.8 and 18.9). Regardless of which model you purchase, centrifugal flotation is considered the gold standard both in the clinic setting and at reference laboratories [4, 7, 8, 22–26]. Centrifugal flotation offers greater recovery of ova [8] as well as greater reliability in the diagnosis of Trichuris vulpis and Giardia lamblia [25], whereas passive fecal flotation may miss as many as 50.5% of infected dogs [27]. Figure 18.6 Passive (gravitational) fecal flotation. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.7 Centrifugal fecal flotation. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.8 Centrifuge model that reflects the swinging bucket style. This style of centrifuge allows you to place the prepared sample into a bucket in the centrifuge, add flotation solution to form a rounded meniscus at the top of the tube, and place a coverslip on the tube. This coverslip will stay in place for the duration of centrifugation. Source: Courtesy of Ryane E. Englar, DVM, DABVP (Canine and Feline Practice). Figure 18.9 Centrifuge model that reflects the fixed‐angle rotor style. Note that because the sample is placed within the centrifuge at an angle, you cannot place a coverslip on the tube during centrifugation. You also cannot form a meniscus at the top of the sample tube with flotation solution or else you risk that the sample will overflow into the centrifuge. Source: Courtesy of Ryane E. Englar, DVM, DABVP (Canine and Feline Practice). For ease of chapter flow, we will review centrifugal flotation first, followed by passive fecal flotation. Note that when performing centrifugal flotation, you can choose to use either a swinging bucket centrifuge or a fixed‐angle centrifuge. We have chosen to outline the procedure for a fixed‐angle centrifuge below. Equipment that is required for fecal flotation with fixed‐angle centrifuge includes: Smaller wax paper cups work best in the authors’ experience, rather than plastic cups. The flexibility of the wax paper will ultimately facilitate pouring the solution into the conical tubes. Plastic cups are more difficult to manipulate during this critical step in the process. Larger sample volumes of feces (e.g., 6–10 g) are acceptable provided that you use a larger volume of flotation solution (e.g., 35 ml). Having two tubes handy also automatically gives you a means by which to balance out the centrifuge. If you only have sufficient sample to fill one tube, then you will have to create a balancer for the centrifuge. You do this by filling another conical tube to the same volume with flotation solution. Figure 18.10 Appropriate sample size for centrifugal fecal flotation. For reference, one Hershey’s Kiss weighs roughly 4.6 g. This amount is less than the size of an adult’s thumb and is roughly the size of a quarter’s surface. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.11 Adding flotation solution to cup containing fecal sample. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Strain the slurry by pouring it through a tea strainer into a clean cup or other suitable container. Use a tongue depressor to agitate the slurry to facilitate its passage through the strainer (see Figures 18.13 and 18.14). Figure 18.12 Mixing fecal sample and flotation solution. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. If you are using woven gauze in lieu of a strainer, unfold the gauze so that you are straining the slurry through a single layer. As the slurry filters through the gauze, apply pressure to the solid matter in the center of the gauze to facilitate the material squishing through. This is very helpful, especially if Sheather’s sugar solution is used. Sheather’s sugar solution is viscous with a high surface tension. As a result, the solution has a propensity to stick to the gauze. A significant portion of your slurry can get “stuck” within the gauze, meaning that you will lose a good amount of your sample to the gauze if you do not gently squeeze the solid matter to help dislodge excess solution. Figure 18.13 Filtering the mixture of feces and fecal flotation solution through a strainer. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.14 Agitating the mixture of feces and fecal flotation solution as it is strained through a filter. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.15 Pouring filtrate into a conical tube. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.16 Capping the conical tube and placing it in the tube rack prior to centrifugation. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. If you had sufficient sample to fill two tubes, then there is no need for an additional balancer. Place both sample tubes opposite each other in the centrifuge and they will effectively balance each other out. If you only had sufficient sample to fill one tube, you need to create a balancer. To do so, fill another conical tube with flotation solution to the same volume as the sample tube and place the balancer tube opposite the sample tube in the centrifuge (see Figure 18.17). Fill the sample tubes with fresh flotation solution to form a slight meniscus (see Figure 18.19). Figure 18.17 Placing conical tubes in centrifuge prior to centrifugation. Note that there are two tubes and that these tubes have been placed opposite each other in the centrifuge to balance each other out. The centrifuge that is pictured here is a swinging bucket type. The authors did not have a comparable picture using the fixed‐angle rotor. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.18 Conical tubes following centrifugation. Note the supernatant and pellet, which are both particularly prominent in the sample on the right. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine. Figure 18.19 Conical tube post‐centrifugation, filled with fresh flotation solution to achieve meniscus. Source: Courtesy of Jeremy Bessett, Inaugural Class of 2023, University of Arizona College of Veterinary Medicine.
18
Fecal Flotation
18.1 Procedural Definition: Whatis This Test About?
18.2 Procedural Purpose: Why Should I Perform This Test?
18.3 Options Available for Fecal Flotation
18.4 Equipment
18.5 Procedural Steps: Fecal Flotation with Fixed‐Angle Centrifuge[1–4, 8, 11, 22, 23, 28]
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