Abstract
The stability of canine urine samples is essential when the samples cannot be analyzed immediately. The objective of this study was to investigate the stability of canine urine samples at room temperature and under refrigerated conditions. Samples from 20 dogs were collected, divided, and stored at 4°C and 20°C. The samples were examined up to 48 h after collection for specific gravity, pH, protein, bilirubin, glucose, ketones, and sediment and at 4 h and 24 h for bacterial growth. Specific gravity and all chemistry parameters were stable for a minimum of 48 h in 90% of samples. The sediment was stable, apart from crystals. The bacterial growth of 3 bacterial species tested in vitro, as well as the clinical samples, was mostly constant over 24 h at the refrigerated temperature. In urine samples stored at room temperature, the total number of aerobic growing bacteria was increasing. The results of our study showed that routinely measured parameters were stable in unpreserved urine for a minimum of 4 h and up to 48 h in most cases. If it is not possible to culture urine immediately, it is recommended that urine samples be stored at 4°C for a period of up to 24 h.
Résumé
La stabilité des échantillons d’urine canins est essentielle lorsque les échantillons ne peuvent être analysés immédiatement. L’objectif de la présente étude était d’examiner la stabilité d’échantillons d’urine canins à température ambiante et réfrigérés. Des échantillons provenant de 20 chiens furent prélevés, divisés et entreposés à 4 °C et 20 °C. Les échantillons furent examinés jusqu’à 48 h après le prélèvement pour la gravité spécifique, le pH, les protéines, la bilirubine, le glucose, les cétones et le sédiment, et à 4 h et 24 h pour la croissance bactérienne. La gravité spécifique et tous les paramètres chimiques étaient stables pour un minimum de 48 h dans 90 % des échantillons. Le sédiment était stable, sauf pour les cristaux. La croissance bactérienne de trois espèces bactériennes testée in vitro, ainsi que dans les échantillons cliniques, était généralement constante sur une période de 24 h à la température de réfrigération. Dans les échantillons d’urine entreposés à la température ambiante, le nombre total de bactérie aérobie augmentait. Les résultats de notre étude démontrent que les paramètres mesurés de routine sont stables dans de l’urine sans agent de préservation pour un minimum de 4 h et jusqu’à 48 h dans la majorité des cas. S’il n’est pas possible de mettre l’urine en culture immédiatement, il est recommandé que les échantillons d’urine soient entreposés à 4 °C pour une période allant jusqu’à 24 h.
(Traduit par Docteur Serge Messier)
Introduction
Maintaining the stability of samples is an essential condition for analyzing laboratory parameters in commercial veterinary laboratories. For urine samples, an immediate investigation is recommended because of the instability of some clinical parameters when samples are transported overnight (1). Routine urine analyses include specific gravity, pH, protein, bilirubin, glucose, ketones, sediment, bacterial differentiation, and quantification. Changes in urine parameters after storage have been studied, especially for pH, bilirubin, glucose, sediment, and bacterial quantification (2–5). Independent of these recommendations, a few studies measured the stability of urine samples in humans and animals under freezing conditions (6–8).
The objective of this study was to investigate the stability of urine parameters at room temperature (20°C) and under refrigerated conditions (4°C). Bacteriology parameters were measured for up to 24 h, while clinical chemistry parameters were measured for up to 48 h, at both temperatures.
Materials and methods
Urine samples were collected from 20 dogs as voided samples between 8:00 and 9:00 a.m. The dogs were either patients or owned by staff of the Small Animal Clinic, University of Goettingen. The dogs were of different breeds and from 1 to 12 y old. Eleven dogs were female and 9 dogs were male.
Samples
After 20 mL of urine were collected from each dog, samples were divided into 20 aliquots, 1 mL in volume each, half of which was used to investigate all nonmicrobiological parameters and the other half for investigating bacteria. Half of the samples from each group was stored at room temperature (20°C) and the other half in the fridge (4°C). The samples were examined immediately after collection and at timepoints 2, 4, 8, 24, and 48 h for clinical chemistry parameters and at 4 and 24 h for bacterial investigation. In addition, an in-vitro validation test was conducted as a control to observe bacterial growth in the urine under defined conditions.
Investigations of nonmicrobiological parameters
Specific gravity was measured using a refractometer (Tooler, Nordhorn, Germany), according to manufacturer’s instructions. As measurements of specific gravity using a refractometer are temperature-dependent, we started by checking the temperature setting for the instrument. Several drops of urine were put on the glass surface of the instrument and the specific gravity was measured immediately. Clinical chemistry parameters were assessed using the Combur 9 Test (Roche Diagnostics, Deutschland, Mannheim, Germany), according to manufacturer’s instructions. Several more drops of urine were transferred to the reaction fields of the dipstick strip and after a reaction time of 30 s, changes in color were assessed. Of the possible parameters of the Combur 9 test, pH, protein, bilirubin, glucose, and ketones were investigated in this study. All sediment examinations were conducted by 1 person (Diplomate European College of Veterinary Clinical Pathology, ECVCP). For that procedure, urine was centrifuged for 5 min at 1000 × g. The supernatant was then removed and 1 drop of the sediment was transferred to a microscope slide. The microscopic examination was done using a low-power (×10) and a high-power (×40) lens. Cells (white and red blood cells, urinary tract epithelia), casts, and crystals were evaluated. The sediment parameters were assessed semiquantitatively based on the number of structures seen per high-power field (0 = 0; < 5 = +; 5 to 10 = ++; > 10 = +++).
Microbiological investigations
Bacterial analysis of urine samples
After the urine samples had incubated for 0, 4, and 24 h at room or refrigerated temperature, a dilution series from 10−1 to 10−8 was plated on solid agar and the plate-count method was used to determine colony-forming units per milliliter (CFU/mL). Urine samples were centrifuged for 5 min at 3000 × g. The sediment was used for bacterial detection on the following media: blood agar and boiled blood agar (Columbia Blood Agar Base) containing a further 0.2% agar-agar (Carl Roth, Karlsruhe, Germany) and 7% defibrinated sheep blood; citrate azide tween carbonate (CATC agar; Sifin Diagnostics, Berlin, Germany) containing 0.1% Tween 80 (Carl Roth); Modified Edwards Medium containing 7% defibrinated sheep blood (Oxoid Thermo Fisher Scientific, Dreieich, Germany); MacConkey Agar (Oxoid Thermo Fisher Scientific); and Brilliance E. coli/coliform Selective Medium (Oxoid Thermo Fisher Scientific). After inoculation, all media were incubated for 24 to 48 h at 37°C under aerobic conditions, except for the boiled blood agar plates, which were incubated under microaerophilic conditions.
The frequency of detected bacteria was divided into absent (−), low numbers (low: 4 to 20 colonies), moderate numbers (mod: 21 to 50 colonies), and high numbers (high: > 51 colonies) of bacteria. Depending on the growth of the microorganisms on the nutrient media, the appropriate nutrient media was inoculated once again from the enriched thioglycolate solution (Oxoid Thermo Fisher Scientific). A 100-μL sample of the urine was transferred in 9 mL of autoclaved thioglycolate solution and incubated at 37°C for 24 h and plated on agar plates. The incubation of the agar plates remained identical to the previous conditions. After the enrichment step, the assessment of bacterial growth was positive (+) or negative (−).
Strain assignment was verified using Gram staining, followed by catalase and oxidase testing, according to standard protocols. Bactident (VWR International, Darmstadt, Germany) was applied according to manufacturer’s instructions to confirm coagulase-positive bacteria. The Streptococcal Grouping Kit (Oxoid Thermo Fisher Scientific) was used to prove streptococcal and enterococcal species. Single grown bacteria species were verified by similarity checks of the 16S rRNA gene sequence against the National Center for Biotechnology Information (NCBI) database using the BLAST search function or substrate utilization methods: API (bioMérieux, Nürtingen, Germany).
In-vitro validation
Bacterial isolates
All isolates used were collected during routine diagnostics of urine samples originating from dogs in the laboratory of the Division of Microbiology and Animal Hygiene in Goettingen, which is accredited according to the standard DIN EN ISO/IEC 17025:2005. The clinical isolates used (Escherichia spp., coagulase-positive Staphylococcus spp., Proteus spp.) are known to be involved in infections of the canine urogenital tract. The identities of the isolates were proven by 16S rRNA analysis (9). Each isolate was stored as a glycerol stock at −80°C. Glycerol stocks were melted on ice and plated onto blood agar plates. Plates were incubated at 37°C for 24 h. A suspension of 1 to 2 colonies of each bacteria species in 10 mL of peptone (Oxoid Thermo Fisher Scientific) was incubated for 24 h at 37°C. Fresh bacteria suspensions were diluted 1:10 with peptone and the number of bacteria was calculated using optical density measurements (Eppendorf BioPhotometer; Eppendorf, Hamburg, Germany).
In-vitro validation tests
Fresh morning urine was collected directly into a sterile urine collection tube from 2 clinically unremarkable dogs. The urine was transported directly to the laboratory, mixed, and filtered through a 0.22-μm filter (Pall, Dreieich, Germany). The sterile filtrated urine was divided into 4 samples. One urine sample was used as a negative control and the other 3 samples were inoculated with 1 of 3 tested bacterial species (Escherichia spp., coagulase-positive Staphylococcus spp., Proteus spp.). To confirm the number of inoculated bacteria, sterile peptone was also inoculated with the 3 bacterial species. Samples were inoculated to a final concentration of approximately 104 cells/mL of each bacterial species. Urine samples were incubated for 0, 4, and 24 h at room temperature (20°C) or refrigerated temperature (4°C). To determine the CFU/mL, the pour plate method was used (ISO 4833-1, 2013). A dilution series from 10−1 to 10−5 of the incubated urine samples was prepared and plated on plate count agar (Carl Roth). The plates were incubated at 30 ± 1°C for 72 h and colonies were enumerated in accordance with DIN EN ISO 7218.
Statistical analysis
For presentation of results, the statistical program GraphPad Prism version 8.00 for Mac and GraphPad Software (La Jolla, California, USA; www.graphpad.com) was used. The statistics were descriptive. Data were presented with line diagrams. Parameter concentrations and time were analyzed with a simple linear regression. The level of significance was P < 0.05.
Results
The specific gravity of urine in the collected samples varied from 1.003 to 1.062 g/mL. In 19 of 20 samples, the specific gravity was stable up to 48 h, independent of the method of storage. In 1 case, there was a slight elevation from 1.034 to 1.036 g/mL after 8 h of storage at room temperature. Under refrigerated conditions, there was also an elevation from 1.034 to 1.036 g/mL after 24 h (Figure 1). Neither elevation was significant (P > 0.05).
Figure 1.
Specific gravity of urine at room temperature and when refrigerated for a period of 48 h. A selection of 5 dogs is shown for better visualization.
The assessment of pH using the Combur 9 strip showed stability up to 48 h in both methods of storage for 18/20 samples (Figure 2). Two samples showed slight elevations after 24 h from pH 5 to 6 in 1 case and from pH 6 to 7 in another case under both storage conditions, which were not significant (P > 0.05). All other parameters assessed by the Combur 9 dipstick strip (bilirubin, glucose, ketones), apart from proteins, showed stable levels over the period of investigation. The assessment of the protein concentration in the urine was stable in 19 of 20 samples. In 1 case, the protein concentration increased from 100 mg/dL up to 500 mg/dL after 8 h under both storage conditions, which was not significant (P > 0.05) (Figure 3).
Figure 2.
Urine pH at room temperature and when refrigerated for a period of 48 h. A selection of 5 dogs is shown for better visualization.
Figure 3.
Protein concentration of urine at room temperature and when refrigerated for a period of 48 h. A selection of 5 dogs is shown for better visualization.
The semiquantitative assessment of urine crystals showed stable crystal quantification up to 4 h. One sample had an elevation at room temperature after 24 h and the same elevation under refrigerator conditions after 8 h. One sample had a slight elevation after 24 h under refrigerated conditions only, another sample at room temperature only, and 1 sample under both conditions. One sample had a slight elevation after 4 h under both conditions. All elevations were not significant (P > 0.05). Apart from this observation, all other parameters that were assessed in the sediment (erythrocytes, leucocytes, epithelial cells, and casts) were stable under both conditions up to 48 h.
Bacterial analysis of urine samples
Ten canine urine samples were analyzed in detail for the occurrence of different bacterial species. The number of CFU/mL was also investigated over an incubation period of 0, 4, and 24 h at room and fridge temperatures.
An overview of the detected bacterial species is provided in Table I. In the urine of dogs No. 2 and 6, no growth of bacteria was detected in either the original samples or after the enrichment step. In the urine samples of the other dogs, Enterobacteriaceae spp. (No. 3, 4, 5, and 9), E. coli (No. 5, 7, and 9), and Enterococcus spp. (No. 1, 8, and 10) were most often detected (Table I).
Table I.
Overview of bacterial species detected in the urine of 10 dogs.
| Dog number | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
|
||||||||||
| Bacterial species | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Enterobacteriaceae spp. | − | − | High | Mod. | High | − | − | − | Mod. | − |
| Escherichia coli | − | − | − | − | High | − | Low | − | Low | − |
| Staphylococcus spp. (coagulase positive) | − | − | − | − | − | − | Mod. | − | Low | − |
| Streptoccus spp. | − | − | − | − | − | − | − | − | Mod.1 | − |
| Enterococcus spp. | + | − | − | − | − | − | − | + | − | Low |
| Bacillus spp. | + | − | − | − | − | − | − | − | − | − |
+ Bacteria species detected in enrichment culture.
− No bacteria detected in original sample and after enrichment step.
Low — low number of bacteria detected in original sample; Mod. — moderate number of bacteria detected in original sample; High — high number of bacteria detected in original sample.
Lancefield group G.
The total number of aerobic growing bacteria over the 24-hour incubation period at room or refrigerated temperatures differed for most tested clinical urine samples below the 1 log10 stage (Figures 4 and 5). In the urine samples incubated at room temperature, those of dogs No. 4 and 9 showed that the number of aerobic bacteria varied more than 1 log10 stage (Figure 4). The detected CFU/mL of dogs No. 1 and 8 also varied more than 1 log10 stage in the urine samples incubated at the refrigerated temperature.
Figure 4.
Number of total aerobic bacteria detected in the urine of 10 dogs incubated at room temperature.
Figure 5.
Measured number of total aerobic bacteria detected in the urine of 10 dogs incubated at refrigerated temperature.
In-vitro validation tests
To confirm the identity of the clinical isolates, the 16S rRNA gene was analyzed. The 16S rRNA gene sequences of the bacterial species were most closely related to sequences of Escherichia coli (sequence similarity 99%), Staphylococcus pseudintermedius (sequence similarity 99%), and Proteus mirabilis (sequence similarity 100%). These 3 bacterial species were therefore chosen for the in-vitro validation test.
No growth of bacteria was observed in the sterile filtered urine samples. To confirm the number of inoculated bacteria, the 3 bacterial species were spiked into sterile filtrated urine and into sterile peptone. E. coli was detected at a concentration of approximately 3.14 × 104 CFU/mL in the urine sample and 2.93 × 104 CFU/mL in peptone. The concentration of detected S. pseudintermedius was 6.00 × 103 CFU/mL in the inoculated urine sample and 5.7 × 103 CFU/mL in inoculated peptone. Proteus mirabilis was detected at a level of 4.43 × 104 CFU/mL in the urine sample and 4.17 × 104 CFU/mL in peptone.
Bacterial growth over time is shown in Figure 6 for incubation at room temperature, while incubation at the refrigerated temperature is shown in Figure 7. A slight increase in the number of bacteria (1.84 × 104 CFU/mL) was observed for S. pseudintermedius over an incubation period of 24 h at room temperature (Figure 6). The total aerobic number of P. mirabilis was increased by 1 log10 (4.31 × 105 CFU/mL) over an incubation time of 24 h. For E. coli, a decrease of 1 log10 (2.91 × 104 CFU/mL) was observed over a time period of 24 h at room temperature. The number of total aerobic bacteria was constant (variation was below 1 log10 stage) over the incubation period of 24 h for all 3 bacterial species tested at the refrigerated temperature (Figure 7).
Figure 6.
Measured number of total aerobic bacteria with standard deviation detected in inoculated urine samples incubated at room temperature.
Figure 7.
Measured number of total aerobic bacteria with standard deviation detected in inoculated urine samples incubated at refrigerated temperature.
Discussion
There is often a delay of up to 24 h between collection and analysis of urine samples at commercial laboratories. It is therefore recommended that urine samples be either analyzed immediately or preserved. Although there are different methods of preservation, the most popular ones are storing at refrigerated and freezing conditions. Among the studies of different preservation methods, 1 study used chlorhexidine-containing tubes and found a good agreement of up to 72 h for nonmicrobiological parameters (10). In another study, post-delivery acidification or alkalization of urine samples did not affect urinary crystals (11). As the interaction between the preservative and some clinical parameters cannot be completely excluded, however, it seemed worthwhile to investigate unpreserved samples.
The results of our study clearly show that specific gravity and the other routinely measured parameters are stable in unpreserved urine for a minimum of 4 h. There was no difference between storage at room temperature or when cooled at 4°C. The specific gravity was stable in our study in 90% of samples, independent of storage method and investigation time. Unfortunately, we could not identify any study that compared specific gravity under different storage conditions over time. One study of canine urine samples found that osmolality was stable in urine samples collected from 5 healthy greyhounds and stored for 7 d at −20°C and −80°C (8). The measurement technique itself seemed to be more significant for reliability. Measuring with a refractometer seems to be better than using a hydrometer or reagent strips (12).
In the present study, bilirubin, glucose, and ketones were stable when stored for up to 48 h at room temperature and at 4°C. Small effects were seen in another study investigating the influence of 24-hour refrigeration on sample stability (13). Two studies showed a greater effect on parameters, such as glucose, in the case of bacterial infection (2,5). In our study, glucose was stable despite bacterial growth and we can therefore conclude that the effect of bacteria on the semiquantitative strip assay seemed to be less significant.
The urine sample from 1 dog showed an increase in protein concentration from 100 mg/dL to 500 mg/dL after 8 h of storage, which was independent of the storage method. As the same dog also had bacteria in its urine, we concluded that the measured protein concentration was due to bacterial growth and the synthesis of ammonia. The association between proteinuria and bacterial growth has been reviewed (14). In addition to proteinuria in cases of urinary tract infection due to an inflammatory process, false positive results of the dipstick as a result of urinary alkalization are also discussed. The source of protein in cases of urinary tract infection is not exactly known, especially in cases without renal involvement (14). False positive results can be caused by urinary alkalization due to urea breakdown by bacteria, which elevates the pH of the urine (15). Some dipsticks, such as the one used in our study, can give false positive results for protein when the urine is basic. The dog with an increased protein concentration also had alkaline urine (pH 9).
Another study investigated urine stored at 6°C and at room temperature (20°C) for the formation of crystals after 6 and 24 h. This study found that crystal formation depended on the duration of storage, with increased crystal formation especially after 24 h and at 6°C. It was recommended that urine sediment be investigated for crystals within 60 min of sample collection (4). We also observed increased crystal concentration under both storage conditions in our study and recommended that sediment be investigated early, although we recommended that it be done before 4 h.
Previous studies showed that most urinary tract infections (UTIs) are caused by bacterial species originating either from the skin of the vulva and prepuce or from feces in the gastrointestinal tract. E. coli, S. pseudintermedicus, Staphylococcus aureus, beta haemolytic Streptococcus spp., Proteus spp., Enterococcus spp., and Klebsiella spp. were detected in 95% or more of urine samples (16–20). We also found these bacterial species most often in our clinical samples. For this reason, we chose 3 clinical isolates typically found in urine samples and involved in UTIs for our in-vitro validation tests. The 16S rRNA analysis demonstrated that the isolates were most closely related to sequences of E. coli (sequence similarity 99%), S. pseudintermedius (sequence similarity 99%), and P. mirabilis (sequence similarity 100%).
Urine samples possess characteristics that have potential antibacterial properties, such as low pH and high concentrations of urea, creatinine, and inorganic ions (21–22). To confirm the number of inoculated bacteria and to exclude an effect of the urine on the growth of bacteria on agar plates, the 3 bacterial species used in our study were inoculated into sterile filtered urine and into sterile peptone at the same concentration. In our study, there was no effect on the total number of aerobic growing bacteria, including E. coli, S. pseudintermedius, or P. mirabilis.
All bacterial species tested in vitro are mesophilic and grow at moderate temperatures (typically from 20°C to 45°C). Therefore, it was not surprising that the CFU/mL of the 3 bacterial species tested in vitro, as well as the clinical samples, were mostly constant over 24 h at the refrigerated temperature. Previous studies also showed that the number of bacteria in urine samples stored at 4°C varied slightly over the time period of up to 72 h (23–25). In contrast, in urine samples that were stored at room temperature, the total number of aerobic growing bacteria increased (24,25). Some of these bacterial species are able to multiply at very high rates, with generation times as low as 30 min (26,27). We also observed more variation in the total aerobic bacteria in urine samples stored at room temperature, but these variations were mostly below 1 log10 levels. Our findings confirm that, if it is not possible to analyze urine samples immediately, it is recommended that they be stored at 4°C for a period of up to 24 h before culture.
This study has a limitation that should be addressed. The observation time of 48 h is considered the usual maximum storage time for urine samples. In special situations, however, if a urine sample was collected on Friday and the analysis could not be done earlier than Monday, the storage time is 72 h, which is a timeframe that should be assessed in future studies.
In conclusion, the results of this study clearly showed that all parameters normally assessed in urine samples are more stable than observed in previous studies. In cases of bacterial infection, both crystal formation and protein may increase after more than 4 h. In general, however, urine is more or less stable for up to 8 h and can be transported to a lab. Cooling conditions may have an effect on bacterial growth, but not on the chemical parameters.
Acknowledgments
The authors thank Desiree Degenhard, Nicole Graefe, and Philipp Katzwinkel (Institute of Veterinary Medicine) for their assistance in sample processing.
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