Am. J. Trop. Med. Hyg., 73(5), 2005, pp. 962-963
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT
ENUMERATING LEPTOSPIRES USING THE COULTER COUNTER
CHRISTINA M. HUMBERD*,
CLINTON K. MURRAY,
SAUNDRA K. STUART,
BARBARA A. REEB, AND
DUANE R. HOSPENTHAL
Infectious Diseases Department, Wilford Hall USAF Medical Center, San Antonio, Texas; the Department of Medicine and Department of Clinical Investigation, Brooke Army Medical Center, Fort Sam Houston, Texas
ABSTRACT
The currently accepted gold standard for enumeration of leptospires is tabulation of organisms using a Petroff-Hausser counting chamber and dark-field microscopy, a technically demanding, time-consuming technique. Quantification of leptospires with a Coulter counter produced reliable and reproducible counts, comparable to the counting chamber. This experiment demonstrates that the faster, less technically demanding Coulter counter may be an alternative to determine numbers of leptospires.
INTRODUCTION
Leptospirosis is a spirochetal infection of worldwide distribution that is increasingly recognized as a cause of significant morbidity and mortality. Leptospirosis research often requires quantification of organisms to produce inoculum or to estimate disease burdens. This enumeration is currently accomplished by directly counting the spirochetes using a Petroff-Hausser counting chamber and dark-field microscopy. Although this process is labor intensive and subject to operator error, it remains the gold standard given a lack of acceptable alternatives.1 Our study investigated an alternative technique, the Coulter counter, for quantification of these organisms.
MATERIALS AND METHODS
Leptospira strains.
Isolates of 11 serovars (10 Leptospira species), originally obtained from the Veterinary Command Food Analysis and Diagnostic Laboratory, Fort Sam Houston, Texas; the American Type Culture Collection (ATCC), Manassas, Virginia; or the laboratory of Dr. David Haake (UCLA), Los Angeles, California, were used in this study (Table 1
). Stock cultures were maintained at room temperature in liquid Ellinghausen McCullough Johnson Harris (EMJH) medium (BD Diagnostics, Sparks, MD). These cultures are subcultured approximately monthly for concurrent use and quarterly for medium-term maintenance.
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TABLE 1 Comparison of automated Coulter counter enumeration of various serovars of Leptospira spp. to manual deternination using dark-field microscopy and a Petroff-Hausser counting chamber
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Inoculum preparation and quantification.
Inoculum cultures were produced by our standard protocol with 1 mL of stock culture placed into 10 mL of fresh EMJH medium and incubated for 7 days at 30°C. On Day 7, counts of each culture were performed with both dark-field microscopy using a counting chamber and with the Coulter counter (Multisizer 3 Coulter Counter, Beckman Coulter, Inc., Fullerton, CA). Manual counting was performed through the use of a Petroff-Hausser counting chamber employing dark-field microscopy. A 1:100 dilution of inoculum cultures were prepared using Hanks Balanced Salt Solution (Krackeler Scientific, Albany, NY). Diluted inoculum samples were then loaded individually by a reader into the counting chamber and enumerated under the dark-field microscope at a magnification of 400x. Each of two independent readers performed manual counts on at least one sample of those strains with more than one counting. Each manual count was performed with a freshly cleaned and loaded chamber. Obtained values were averaged to minimize inter-observer variation. Automated counting using the Coulter counter was performed using the same diluent, initially at dilutions of 1:10, 1:100, 1:1,000, and 1:10,000 to assess the optimal concentration of leptospires that the machine could reproducibly produce results from. The most consistent automated results were obtained with the 1:1,000 dilution (data not shown) and thus this dilution was used for the remainder of the study.
Statistical analysis.
Study of five isolates was performed in triplicate to assess reproducibility. A two-factor ANOVA analysis followed by the two-tailed t test was performed to assess for significant differences in the means from either method (significance P > 0.05).
RESULTS
Counting of leptospires from five common species; serovars Butembo, Fortbragg, Borincana, Patoc, and Icterohaemorrhagiae was performed three times by each method and the results compared by statistical methods described (Table 1
). Only serovar Icterohaemorrhagiae had a statistically significant difference in leptospire counts between the two techniques. Similar results were noted with serovars in which counts were performed less than three times each by these methodologies. In all tests, the two techniques produced counts within a half log of each other.
DISCUSSION
Leptospirosis studies are being published with increasing frequency, necessitating the development of newer laboratory techniques to aid in these investigations. The current dependence on manual counting of these spirochetes with the Petroff-Hausser counting chamber using dark-field microscopy significantly slows research and likely increases the possibility of operator error. In this study, the Coulter counter was found to be a useful device for leptospirosis enumeration that appears to provide equivalent results to those obtained using the current counting chamber method.
There have been few studies published on techniques for enumeration of leptospires. The most widely used method is microscopic counting using a Petroff-Hausser counting chamber and dark-field microscopy. This technique is time and labor intensive.2 Studies have evaluated the use of plate counts to determine colony-forming units, but this technique requires a long incubation period, from 12–14 days.3 In addition, counts obtained through the pour-plate technique represent viable count, which may be different from the total count derived from dark-field microscopy or the Coulter counter. Additionally, there are methods for indirect quantification, measuring the turbidity of the culture media. However, because of relatively low optical density, currently available spectrophotometers are too insensitive to measure concentrations of 108 or less and are thus limited in their utility.4
The Coulter counter has been in use for bacterial cell counting as documented in the literature since 1966. Several studies have demonstrated its utility for counting various bacterial organisms from Bacillus species to cocci.5,6 In one later study, a direct linear relationship was demonstrated when counts of Escherichia coli, Staphylococcus aureus, and Streptococcus faecalis obtained by the Coulter counter were compared with those obtained by the pour plate technique. The ease and rapidity of counting with the Coulter was felt to make this technique superior to the pour plate method.7
Limitations of this study include the lack of evaluation of this technique on different types of cultures or directly from infected tissues or fluids. Future work should include a more extensive evaluation of the Coulter counter for leptospirosis enumeration, to include from other sources such as tissue culture and directly from experimentally infected animals. In summary, the study of leptospirosis is complicated by the lack of efficient techniques for quantification and this study demonstrates that the Coulter counter may provide a useful alternative.
Received April 26, 2005.
Accepted for publication May 27, 2005.
Disclaimer: The views expressed herein are those of the authors and do not reflect the official policy or position of the Department of the Army, the Department of the Air Force, Department of Defense, or the U.S. Government. The authors are employees of the U.S. government. This work was prepared as part of their official duties and, as such, there is no copyright to be transferred.
* Address correspondence to Christina M. Humberd, USAF, MC Infectious Disease (759 MDOS/MMII), Wilford Hall Medical Center, 2200 Bergquist Drive, Suite 1, Lackland AFB, TX 78236-9908. E-mail: Christina.Humberd{at}lackland.af.mil 
Authors’ addresses: MAJ Clinton K. Murray, MC, USA, Infectious Disease (MCHE-MDI), Brooke Army Medical Center, 3851 Roger Brooke Drive, Fort Sam Houston, TX 78234, Telephone: (210) 916-4355, Fax: (210) 916-0388, E-mail: Clinton.Murray{at}amedd.army.mil. Capt Christina M. Humberd, USAF, MC, Infectious Disease (759 MDOS/MMII), Wilford Hall Medical Center, 2200 Bergquist Drive, Suite 1, Lackland AFB, TX 78236-9908, Telephone: (210) 292-6726, Fax: (210) 292-3740, E-mail: Christina.Humberd{at}lackland.af.mil. Saundra K. Stuart and Barbara A. Reeb, Department of Clinical Investigation (MCHE-DCI), Brooke Army Medical Center, 3851 Roger Brooke Drive, Fort Sam Houston, TX 78234, Telephone: (210) 916-4355, Fax: (210) 916-0388. LTC Duane R. Hospenthal, MC, USA, Infectious Disease (MCHE-MDI), Brooke Army Medical Center, 3851 Roger Brooke Drive, Fort Sam Houston, TX 78234, Telephone: (210) 916-4355, Fax: (210) 916-0388.
Reprint requests: MAJ Clinton K. Murray, MC, USA, Infectious Disease (MCHE-MDI), Brooke Army Medical Center, 3851 Roger Brooke Drive, Fort Sam Houston, TX 78234, Telephone: (210) 916-4355, Fax: (210) 916-0388, E-mail: Clinton.Murray{at}amedd.army.mil.
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