Claims:
1. A method for non-discriminative microbe consolidation/separation by using electrokinetic separation device with a separation pathway, comprises: introducing a sample into a sample buffer to make a sample solution; introducing said sample solution into the separation pathway with running buffer containing a cationic agent; introducing a segment of solution containing a blocking agent in to the separation pathway; performing an electrokinetic separation.
2. A method for non-discriminative microbe presence/absence testing (sterility testing) by using electrokinetic separation device, comprises: introducing a sample into a sample buffer to make a sample solution; introducing said sample solution into the separation pathway with running buffer containing a cationic agent; introducing a segment of solution containing a blocking agent; performing an electrokinetic separation; detecting the presence/absence of the microbe(s).
3. A method as claim 1, where the cationic agent is a single tailed cationic surfactant.
4. A method as claim 4, where the single tailed cationic surfactant is CTAB.
5. A method as claim 5, where the concentration of CTAB ranges from 0.5 mg/ml to 2 mg/ml.
6. A method as claim 1, where the cationic agent is a double tailed cationic surfactant.
7. A method as claim 1, where the cationic agent is a geminal di-cationic liquid/salt.
8. A method as claim 1, where the blocking agent is a nutrient broth.
9. A method as claim 1, where the blocking agent is a peptide(s).
10. A method as claim 1, where the blocking agent is a zwitterion.
11. A method as claim 1 , where the blocking agent is a zwitterionic detergent.
12. A method as claim 11, where the zwitterionic detergent is caprylyl sulfobetaine.
13. A kit for the performance of the rapid non-discriminative microbe consolidation/separation, comprises: a cationic agent; a blocking agent.
14. A kit as claim 13, where the cationic agent is a solute in a running buffer.
15. A kit as claim 13, where the blocking agent is a solute in a solution.
16. A kit as claim 13, where the cationic agent is a single tailed cationic surfactant.
17. A kit as claim 13, where the single tailed cationic surfactant is CTAB.
18. A kit as claim 14, where the cationic agent is CTAB in the concentration ranges from 0.5 mg/ml to 2 mg/ml.
19. A kit as claim 13, where the cationic agent is a double tailed cationic surfactant.
20. A kit as claim 13, where the cationic agent is a geminal di-cationic liquid/salt.
21. A kit as claim 13, where the blocking agent is a nutrient broth.
22. A kit as claim 13, where the blocking agent is a peptide.
23. A kit as claim 13, where the blocking agent is a zwitterion.
24. A kit as claim 13, where the blocking agent is a zwitterionic detergent.
25. A kit as claim 13, where the zwitterionic detergent is caprylyl sulfobetaine.
26. A kit of claim 14, where the running buffer solution further comprises of TRIS and citric acid.
27. A kit of claim 13, further comprises a EOF marker.
28. A kit as claim 27, where the EOF marker is a solute in a solution.
29. A kit as claim 27, where the EOF marker is DMSO.
30. A kit of claim 13, further comprises a fluorescent microbe dye.
31. A kit as claim 30, where the fluorescent microbe dye is a solute in a solution.
32. A kit as claim 30, where the fluorescent microbe dye is a Baclight fluorescent dye. |
NON-DISCRIMINATIVE MICROBE SEPARATION AND TESTING
FIELD OF THE INVENTION
This invention relates to separations-based techniques for an efficient/effective method of providing a simple, rapid, and binary (yes/no) answer in regard to the presence/absence of any/all microorganisms.
BACKGROUND OF THE INVENTION
Testing for the presence of microbes, whether they are bacteria, fungi, or even viruses, in laboratory samples is an important and necessary procedure for many areas of science. The food industry must be sure that there is minimal microbial contamination in products, especially contamination that could cause consumers illness upon ingestion. Microorganism testing is important in the pharmaceutical industry as well, as it produces a large number of medicinal products for consumer use. The health care industry must be very careful that tissues or other important biological samples (i.e., blood or plasma) are not infected with microbial agents. A transplant of these materials to an otherwise healthy patient could prove disastrous. It is important for hospitals to have the ability to diagnose bacteremia or urinary tract infections (UTIs) and to do so quickly; a quicker diagnosis leads to faster treatment and recovery. Furthermore, it is essential that the large amount of water processed by treatment plants is suitable for use by the general public, and that
special sterile water/aqueous solution samples used in medicinal and microbiological research are indeed microbe free.
There has been growing interest in separations-based techniques for the identification and characterization of microorganisms because of the versatility, selectivity, sensitivity, and short analysis times of these methods. A related area of analysis that is scientifically and commercially important is the determination of the presence or complete absence of microbes (in essence, a test for sample sterility). In such a test, it is not of immediate importance to identify a particular microorganism, but rather, to know with a high degree of certainty whether any microbe(s) is (are) present.
There is a pressing need for a rapid test that is capable of providing a binary answer regarding the presence/absence of a wide variety of microorganisms. Culture methods and modifications of them are still the universally accepted procedures for determining microbial contamination, but these types of tests suffer from long analysis times. Other techniques such as hybridization, amplification, and immunoassays can considerably reduce the time required for microbial analysis; however, they all have specific drawbacks as well. Some of these techniques require considerable training and expertise to perform. They do provide a high level of specificity, in terms of identifying species, but this is not always desired or needed for a simple "sterility" test. The method presented here is capable of providing a quick (a few minutes) answer regarding the presence or absence of microorganisms. It is also applicable to a variety of different bacteria, even to samples containing a variety of different species. In this manner it is able to utilize the best elements of other available tests. It can potentially be used to diagnose bacteremia or UTIs; these are cases where a quick answer can be much more
beneficial than a detailed answer (providing genus, species, and strain information) which may consume precious extra time.
A number of different choices are available when testing for microorganisms, each with its own advantages and disadvantages. Standard culture methods can provide accurate information about the presence and number of bacteria by using serial dilutions and colony counting, yet they are very time-consuming and do not count dead microorganisms or microorganisms that do not grow in a particular medium. Molecular methods can reduce this time requirement considerably and can even provide information about the very genetic makeup of the bacteria. However, to detect general contamination, this degree of specificity is not needed, and indeed the high degree of specificity of these methods will result in false negatives as other microorganisms go undetected. Another example is a case where the source of contamination is not known; it is only known that the sample has been in an environment where contamination from any number of bacteria is likely. Tests that are developed for specific bacteria will not always give meaningful information in such a case, unless by chance the sample was contaminated with the same microorganism the test was designed for.
There is a need for an efficient/effective method capable of providing a simple, rapid, and binary (yes/no) answer in regard to the presence/absence of any/all microorganisms. Such a test will greatly reduce the time requirements in cases where specificity is not needed, and also when it is used as a first-step analysis where specificity is required to ensure that more complex or time-consuming tests are not performed on samples that have not been contaminated with bacteria in the first place. The test should
combine the reduced analysis time of molecular techniques, possibly even reducing them further, and the broad applicability of culture techniques.
Analytical techniques based on electrokinetic separation methods, such as capillary electrophoresis (CE) and related microfluidic approaches have been used recently to address some of the problems associated with microbial detection and identification methods. These are-attractive techniques because of their fast analysis times and very small sample requirements (which often is the case with microbial samples). However, a majority of the work with microorganisms and CE has focused on certain kinds of bacteria, fungi and/or viruses. There have been very few reports on the identification of the presence or complete absence of a large number of diverse microorganisms by electrokinetic methods, in essence a sterility test.
SUMMARY OF THE INVENTION
Innovative electrokinetic methods and kits for rapid non-discriminative microbe separation and testing are described in this invention. These electrokinetic methods promote the consolidation of all microbe types into a single zone (peak) which is separated from the electroosmotic flow (EOF) front and any other interfering molecular constituents. It can be accomplished using a segment of dilute cationic agent, such as cetyltrimethylammonium bromide (CTAB), which serves to temporarily reverse the migration direction of the microbes, and another segment of solution containing a "blocking agent", such as zwitterions, which serves to stop the microbes' migration and
focus them into a narrow zone. The kits of this invention are for easy performance of the methods by providing certain combination of the reagents in a convenient package.
This approach is effective for a broad spectrum of bacteria, fungi and virus. By using the methods and kits in this invention, the separation and testing of the presence/absence of a microbe can be accomplished in less than 10 minutes.
In particular, the invention of this application is a method for non-discriminative microbe consolidation/separation by using electrokinetic separation device with a separation pathway. The method of the present invention comprises introducing a sample into a sample buffer to make a sample solution, injecting the sample solution into the separation pathway with running buffer containing a cationic agent, then injecting a segment of solution containing a blocking agent in to the separation pathway and then performing electrokinetic separation. It further provides a method for non-discriminative microbe presence/absence testing (sterility testing) by using electrokinetic separation device, comprising the steps of introducing a sample into a sample buffer to make a sample solution, injecting said sample solution into the separation pathway with running buffer containing a cationic agent, then injecting a segment of solution containing a blocking agent, and then performing electrokinetic separation with the testing a microbe.
In some embodiments, the invention uses a geminal di-cationic liquid/salt as the cationic agent. In some other embodiments, the invention uses a single or double tailed cationic surfactant, such as CTAB as the cationic agent. Preferably, the concentration of CTAB ranges from 0.5 mg/ml to 2 mg/ml.
In some embodiments, the blocking agents are the following agents: a nutrient broth or other solution with a peptide, peptide digest, a zwitterion, or zwitterionic detergent, such as caprylyl sulfobetaine.
This invention also provides the kit for the performance of the method for rapid non-discriminative microbe consolidation/separation, including a cationic agent and a blocking agent, either in dry components or in solutions with suitable concentration. If the agents are in solution, for example, for the cationic agent CTAB solution, the concentration is in the range from 0.5 mg/ml to 2 mg/ml.
The kit embodiments of this invention may further including TRIS and citric acid in the running buffer, an EOF marker, such as DMSO, and a fluorescent microbe dye, such as the Baclight dye, either in dry components or in a solution with suitable concentration.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates the supposed mechanism of the electrokinetic method.
Figure 2 illustrates the unique arrangement by the method of this invention. "A" is the injected sample solution; "B" is the running buffer with the cationic agent, in this case the CTAB; "C" is the segment of the blocking agent solution. The overall flow is in the EOF direction to the anode.
Figure 3 illustrates the typical electropherogram, in which the first peak is the detector response to the EOF marker and the later sharp peak is the detector response to the microbe(s).
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to a procedure for issuing a binary answer to the presence/absence of a broad array of microorganisms in a single sample by using an electrokinetic method in conjunction with cationic agents and blocking agents. This method is capable of discerning the presence of microbes in a very short time. It can detect the presence of a single cell (therefore the absence of all cells, i.e., a sterile sample). The best elements of culture techniques (broad applicability) and molecular techniques (fast analysis times) are employed in the invention. While it is not capable of the specificity of molecular techniques in terms of identifying bacteria at the species or strain level, it is useful as a stand-alone sterility test or as a quick first-step analysis when specificity is not needed.
For more sensitive and effective testing, the bacteria should be compressed at a point inside the capillary. This results in all of the aggregated microbes migrating together at the same velocity, as long as all other forces inside the capillary (e.g., turbulent flow, thermal effects, etc.) were insufficient to break them apart. For example, even if multiple bacterial species are present, it does not pose a problem since interspecies aggregation is not uncommon.
It is well-known that acidic conditions can induce aggregation in bacteria, and initial experiments with a method developed using pH boundaries had some success. This method worked well for some bacteria but not for others. Mainly Gram-positive bacteria (e.g., Bacillus subtilis, L. innocua, etc.) aggregate under acidic conditions. Such a procedure can be useful as a secondary analysis for the identification of Gram-positive bacteria. To accomplish the main goal, however, a method that was capable of causing a wider number of different bacteria to associate well away from the EOF front has to be discovered.
The addition of dilute cationic agent, such as CTAB to the running buffer caused some bacteria to undergo aggregation. The concentration of CTAB, when used as an agent, is low (~1 mg/mL) and does not lyse the cells. The introduction of a blocking agent, for example, by placing a segment of nutrient broth after the injection of the sample, or by adding nutrient broth to the sample solution caused a large number of bacteria to aggregate.
It should be noted that this aggregation is not a coprecipitation process; no precipitate forms when only CTAB and nutrient broth are combined. It appeared that combination of CTAB and nutrient broth had a synergistic effect toward consolidation of all types of bacteria. Also it was not sensitive to the effects of microbial electrophoretic heterogeneity. This invention provides an electrokinetic method utilizing this effect to allow the microbes, migrating as a single band, to distance themselves from the EOF, to distinguish them from contaminants appearing there.
The general process is set up in the following manner. The channel or capillary is rinsed with water and base and then filled with run buffer containing cationic agent, such as CTAB in an appropriate concentration. The sample specimen is suspended in a solution and centrifuged. The microbe concentrated as the sample is then introduced into a sample buffer (it does not contain CTAB) to make a sample solution. The sample solution is then injected. Later, a segment of blocking agent, such as nutrient broth (which also does not contain CTAB) is placed in the pathway. The dissolved CTAB residing in front of the bacteria (on the anodic side) migrates toward the cathode when the voltage is applied. As it passes through the sample zone it carries the bacteria with it. The electroosmotic flow is reversed under these conditions, and it flows toward the anode, as does the segment of nutrient broth that was injected. The combination of the anodic movement of the nutrient broth segment and the cathodic movement of the bacteria allows them to converge at a point between the two zones in the capillary. The bacteria begin to aggregate and eventually form a large macroparticle. As the macroparticle forms, it quickly loses mobility, and from that point on it migrates in the anodic direction while residing in the blocking segment. The arrangement by this process is illustrated in Figure 2.
It is believed that the mechanism of this inventive method is as follows. Under the condition of electrokinetic separation, the cationic agent(s), such as CTAB moves into the region containing the microbes, since it had a current motion counter to the electroosmotic flow. When CTAB reaches the microbial section, it will interaction with the negatively charged microbes' membrane to reduce or stop their movement along with the EOF. This mechanism is illustrated in Figure 1. The uncoated pathways surface (glass,
silicone plastic and other suitable material for the separation pathway) "1" is with negative charge in most of the conditions; the reversed surface charge "2" is due to the cationic surfactant and its associated negative charged diffusion layer; the EOF direction "3" is to the anode, which is induced by the negative charged diffusion layer; as "4", the cationic surfactant affiliating with the negatively charged surface of the microbes, and therefore give them a net positive charge; the microbes' movement "5" is a counter current relative to the EOF current, which is due to the net positive charge of the microbes.
The suitable cationic agents include single or double tailed cationic surfactants and geminal di-cationic liquids/salts represented in the following charts:
Imidazolium-based Dicationic Ionic Liquids
λ = Bf. M k . IiYi . M < : \ = Br . N l f;f. BF 4 " . PIY n - 3. C ?{mim);-A Ii - .). C'.(ιn;im) 2 -λ
Pyrrolidiniuin-based Dicationic Ionic LiciitUh
A sharp peak will appear; when the microbes' counter current movement is hindered by a "blocking agent", which serves as a screen segment for the aggregated microbe particles to prevent them migrate out of the blocking region. The blocking agent should be interactive with the aggregated microbe particles. Proper blocking agents can be a nutrient broth, peptides, zwitterions, or a zwitterionic detergent.
Similar results may be generated by putting the sample into a solution with the blocking agent, or by placing a segment of block agent into the separation pathway after
the injection of the sample solution. The microbial typically has migration times longer than that of the EOF.
Microbes are known to be negatively charged at conditions similar to those at which these experiments were performed. Under normal circumstances this type of electropherogram (a peak with a longer migration time than the EOF) is further evidence of the negative charge of the bacteria; however, under conditions where both the electroosmotic flow and polarity are reversed (as in these experiments), it indicates exactly the opposite. It was apparent that the dilute cationic agent, such as CTAB, has a significant effect on the mobility of the bacteria.
Further investigation has been performed to examine the exact effect of CTAB on the bacteria. The capillary was filled with buffer containing CTAB, then an initial injection of buffer lacking CTAB was made, and finally a sample consisting of bacteria and an EOF marker was injected. The migration time of the bacteria in this instance was the same as that of the EOF marker. This was sufficient to show that the CTAB in the region adjacent to the bacteria (on the anodic side) was responsible for imparting some positive charge to their surface. Due to the opposite direction of migration of the bacteria and the EOF (which carried the nutrient broth), it was thought that separating the two regions, with the bacterial sample on the anodic side of the capillary and the blocking segment on the cathodic side, allows them to converge in an area between the two injected segments. Also, this could solve the problem of any interference that could be present near the EOF front since the CTAB is capable of carrying the bacteria well away from that region.
The system used in this invention is fairly complex. Not only are there the standard factors of pH, ionic strength, and additive concentration, which are commonly optimized for all electrokinetic experiments, but also there are other factors that must be considered. The sample injection amounts were optimized in an effort to introduce as much sample into the pathway as possible. This was done by increasing either the injection time or the inner diameter of the separation pathway. Introduction of larger amounts of sample could prove useful when dealing with very dilute samples.
Three different pHs were tested using this method: 4, 7, and 9. Each pH yields a very sharp single peak for the bacteria (E. coli); however, pH 7 seemed to consistently give peaks with a higher absorbance. The peaks do not tend toward longer migration times as the pH is lowered. This is because of the complex nature of the dependence of electroosmotic flow on the cationic agent concentration. A combination of TRJS and citric acid at fairly low concentrations was used as the running buffer for this system.
There is a narrow window of workable ionic strengths as compared to pH. A single peak is obtained at 5 times the normal concentration of TRIS/citrate used; however, it is slightly widened and skewed. The peak converts back to its normal shape upon addition of more CTAB. However, a further increase to 10 times the normal concentration of TRIS/citrate completely destroys the single peak, and it cannot be reconstituted even if further amounts of CTAB are added. A solution consisting of only CTAB and water can be used to obtain similar results; therefore, only the upper limit of ionic strength significantly affects the peak number and shape.
When the run buffer concentration in these experiments is varied, it is imperative that the sample buffer be of the same ionic strength. If the sample buffer is significantly higher or lower in ionic strength than the surrounding run buffer, an adequate peak is not obtained (i.e., multiple peaks or inconsistent migration times occur). In the case where the sample buffer is of lower concentration than the surrounding run buffer, multiple peaks are obtained. If the sample is of higher ionic strength than the surrounding run buffer, the bacteria again become trapped in the neutral zone well before they reach the blocking segment. It is reasonable to assume that CTAB has an effect on the surface of the bacteria since the mobility of the bacteria has been shown to be affected by it. There are a number of counterions around any charged particle in solution, and the density of the ions depends on the ionic strength of the solution. It is believed that, at higher ionic strengths, when the diffuse layer becomes more compact, it may be harder for the CTAB to reach the surface of the bacteria. Thus, the CTAB may not be able to affect the surface of the bacteria in the way it does at lower ionic strengths. An alternate explanation is that when this ionic layer becomes compact the bacteria tend to aggregate slightly. The larger aggregates, as opposed to mostly single bacteria at low ionic strengths, may not be affected as greatly by the cationic agent.
The concentration of CTAB in the run buffer was varied to study its effect on the system. The optimal concentration of CTAB is between 1 and 2 mg/mL; the peak height drops considerably if the concentration is raised. This phenomenon may be possibly due to the lyses of cells as the concentration of CTAB increases. The highest signal obtained while still exhibiting a single peak is most desirable, since a major goal is detecting bacteria at very low levels. The sharp single peak that is usually obtained breaks apart at
CTAB levels of ~0.5 mg/mL. The workable concentration for other cationic surfactants should be in the range between their respective critical micelle concentration (CMC) as the upper limit and the need concentration to give a double-layer coverage of the inner surface of the separation pathway and the outer surface of the microbial membranes, as the lower limit. For the di-cationic liquid, the concentration can be much higher, because the do not tend to lyse cells.
It is better to obtain as large of a signal as possible by optimizing the conditions. This allows detection of microbes at the very low levels. In addition, it also helps to compensate for the low number of cells injected and detected due to the inherently low volume of the capillaries used. Therefore, experiments were undertaken to maximize the bacterial injection volume. This was accomplished by use of both longer capillaries and larger inner diameter capillaries. The longer capillaries were used to increase the length of the injected bacteria zone.
Filling the capillary with too long of a sample segment adversely affects the EOF since the sample does not contain CTAB, and CTAB is responsible for coating the capillary and reversing the EOF. If the sample zone gets too large, the amount of CTAB present to reverse the flow is insufficient and it will lead to prohibitively long migration times. Use of longer separation pathways helps to minimize this effect. More of the remaining capillary is filled with CTAB for two equal-length injections when a longer capillary is used. The longer capillary also affords the bacteria more time to reach the blocking segment. The use of larger inner diameter capillaries allows larger volumes of the bacterial sample solution to be injected for equal injection lengths.
A comparison was made between the use of a 30 cm separation pathways and a 60 cm separation pathways. The peak shape is normal for injections of 6 and 9 s (at 0.5 psi) for the 30 cm capillary, but the injection of 12 s (at 0.5 psi) shows multiple peaks. Experiments have shown that cationic agent cannot be present in the segment containing bacteria and also that cationic agent is responsible for moving the bacteria in the cathodic direction; therefore, the distance over which the cationic agent must carry the bacteria becomes increasingly large as the bacteria are injected over longer time periods. It seems that a limit is reached at which the cationic agent cannot carry all of the bacteria in the cationic agent free sample segment back to the interface of the blocking segment. In this case multiple peaks are observed. A 60 cm capillary was used in an attempt to give the cationic agent additional time to carry the bacteria back to the nutrient segment and help unify the multiple peaks obtained for long injection times.
The injection pressure was identical to that of the 30 cm capillary (0.5 psi), but the length of time for each injection was doubled; this was done so that direct comparisons could be made between capillaries. The injections of 12 and 18 s on the 60 cm capillary, which correspond to the 6 and 9 s injections on the 30 cm one, both gave reasonable peaks. However, similar to the 30 cm capillary, additional peaks appeared when the injection time was increased to 24 s (12 s on the 30 cm capillary). Therefore, the additional time afforded to the CTAB did not improve the microbial peak.
Increasing the inner diameter of the capillary proved to be much more useful for increasing the overall number of bacteria and volume of solution injected. Two different inner diameter (i.d.) capillaries (100 and 200 mm, both 30 cm in length) were used for these experiments. The concentration of microbe injected was identical in each
experiment as were the lengths (not in seconds but in millimeters) of the injected spacer, nutrient segment, and microbial sample segment. The time and pressures were adjusted for injections made on the 200 mm i.d. capillary to compensate for the larger flux of materials and to give injection lengths similar to those of the 100 mm i.d. capillary. Both experiments produce sharp microbial peaks.
However, it is clear that a much larger peak is obtained when using the 200 mm i.d. capillary. The doubling of the diameter leads to a 4-fold increase of the volume injected if the injection length (again in millimeters, not seconds) is held constant. The increased number of bacteria injected in this larger capillary most likely leads to the larger signal, since they are compacted into the same length as in the smaller diameter capillary. In addition there is an added benefit of increased (double) path length for detection when using the larger inner diameter; this also adds to a larger signal in accordance with Beer's law.
There are some points that should be mentioned with the use of larger capillaries. For instance, molecular species are broadened significantly in 200 mm capillaries (compared to 100 mm), due to increased Joule heating, siphoning, and other effects. This is not the case with this microorganism procedure. Upon aggregation they travel as a single macroparticle and cannot be separated by the forces of thermal mixing or other dispersive actions within the capillary. Theoretically, even larger diameter capillaries could be used; however, practical concerns become a problem. For instance, these larger i.d. capillaries may be more brittle and prone to breakage. Also, most commercial CE instruments are only capable of accepting common outer diameters of the capillary
(usually 360 mm). This indicates that another instrument should be developed that works optimally for electrokinetic sterility testing.
Experiments were also performed to determine the maximum injection length for a capillary that is 30 cm long and has an inner diameter of 200 mm. The injection lengths used ranged in time from 4 to 7 s (at 0.2 psi). As is expected, the peak height increases with the injection time, due to the greater number of bacteria being injected. The migration time also increases as the injection time increases; this is a result of the length of the CTAB-free sample zone. As mentioned previously, the reversed flow of the EOF is dependent on the amount of CTAB in the capillary. As the concentration of CTAB decreases, the velocity of the reversed EOF decreases as well, resulting in longer migration times. At a 7.0 s injection, the peak begins to broaden and split slightly. This is roughly the limit of the injection for a capillary of this diameter. This 6.0-7.0 s maximum injection time for the 200 mm i.d. capillary is of particular interest. This time corresponds to an injection length of 43-50 mm and an injection volume of 1360-1580 nL (using the Poiseuille equation). This is nearly identical to the maximum injection length of 40-53 mm for a 100 mm i.d. capillary. However, the volume injected is of course much lower for the 100 mm i.d. capillary at 317^4-23 nL. Though there is a limitation as far as the injected sample length is concerned, the injected sample volume (and hence number of bacteria) can be increased significantly by simply increasing the inner diameter of the capillary. Fortunately, doing so does not adversely affect the results.
In the application of the methods of this invention, an EOF marker may be used to indicate the front of the EOF of the experiments, which may serve as the monitoring
starting time for the testing. Suitable EOF markers includes DMSO, thiourea, acetone, acrylamide, nitromethane, propanal, and acetic acid.
A majority of the microorganisms in this study fall into the biosafety level one category, and standard microbiological practices can be employed in their use. Two microorganisms used in this study (Pseudomonas aeruginosa and S. aureus) fall into the biosafety level two category, and when handling these microorganisms, extra precautions should be used. This includes using extreme caution when handling sharps or needles contaminated by the microorganism. Procedures that produce aerosols or have the potential to splash should be done in biosaftey cabinets or safety centrifuge cups. Splash shields, face protection, gowns, and gloves are recommended. Waste decontamination must be available as well.
These methods apply broadly to many microorganisms. Regardless of whether the sample contains many bacteria, fungi, viruses, or just a single species, the same sharp peak will occur at the same point in the capillary in each instance. Also note that it takes less than 10 min for any combined microbial band to migrate to the detector. Hence, it could be used to obtain rapid information as to the presence or absence of microorganisms in a sample. It could also be multiplexed for high-throughput analysis.
This invention provides the method capable of giving a rapid, unambiguous answer as to the presence/absence of microbes. Methods generating a single peak at the desired time, regardless of the microbial species, its heterogeneity, or the number of different species, and a kit for the application of the method are presented in this invention. Capillary electrophoresis is one type of eletrokinetic method for use in this
invention. These and other aspects of the present invention may by more fully undertood by reference to the following examples.
Example 1: General Microbe Testing
Materials: Tris(hydroxymethyl)arninomethane (TRIS), sodium hydroxide, hydrochloric acid, and cetyltrimethylammonium bromide (CTAB) were all purchased from Aldrich (Milwaukee, WI). Citric acid was obtained from Fisher Scientific. Dimethyl sulfoxide (DMSO) was a product of EM Science (Gibbstown, NJ). Luria broth was obtained from Sigma (St. Louis, MO). Brain heart infusion and nutrient broths were obtained from Difco Laboratories (Franklin Lakes, NJ.). BacLight fluorescent dye was obtained from Molecular Probes, Inc. (Eugene, OR). Uncoated fused silica capillaries with inner diameters of 100, 150, and 200 mm and outer diameters of 365 mm were obtained from Polymicro Technologies (Phoenix, AZ). Escherichia coli (ATCC no. 10798), Salmonella subterreanea (ATCC no. BAA-836), Listeria innocua (ATCC no. 33090), Brevibacterium tapei (ATCC no. 13744), Corynebacterium acetoacidophilum (ATCC no. 13870), Aerococcus viridans (ATCC no. 1 1563), Pseudomonas flourescens (ATCC no. 11150), Escherichia blattae (ATCC no. 29907), and Staphylococcus aureus (ATCC no. 10390) were all obtained from the American Type Culture Collection (Manassas, VA).
Methods: All bacteria were grown according to the directions supplied by the manufacturer. The microorganisms were initially grown in liquid broth, then plated on agar plates, and stored under refrigeration until needed. When used for experiments, a single colony was taken from the agar plate and again grown in a liquid medium. The
cells were harvested for use when in the stationary phase of growth when the concentration was -108 CFU/mL. The microorganisms were centrifuged, the excess broth was removed, and the microorganisms were washed with working concentrations of the TRIS/citric acid buffer (pH 7) once, then recentrifuged and decanted, and finally suspended in a working concentration of TRIS/citric acid buffer of the same volume as the broth that was originally removed. The final concentration of the cells was ~10 8 CFU/mL. These were then used as samples for analysis. This was done each day to produce new samples.
The following procedure for staining the microorganisms with BacLight dye was used when LIF detection was necessary. Briefly, a 1 rnM stock solution of BacLight Green dye was prepared in DMSO. A working solution of 100 mM was then made by adding 2 mL of the 1 mM stock solution to 18 mL of DMSO. The cells were stained by adding 1 mL of the working dye solution/mL of bacteria solution, and then incubated at room temperature for at least 15 min. With UV detections, the signal for the bacteria varied, but generally the lowest number of microbes detectable was around 50-200 entities.
The separation pathways used in these experiments varied in length from 30 to 60 cm (20 and 50 cm to the detector, respectively). The inner diameters also varied from 100 to 500 um. When a capillary was first used, it was rinsed with water for 30 s, 1 N NaOH for 5 min, and the running buffer for 2 min. Prior to each run the 100 mm i.d., 30 cm capillary was rinsed with water for 1 min, base for 1 min, and buffer for 1 min at 10 psi. The 200 mm i.d., 30 cm capillary was rinsed with water for 10 s, base for 10 s, and buffer for 10 s at 5 psi. When 60 cm length capillaries were used, the rinse time was doubled
while the pressures remained the same. DMSO was used as a neutral marker at a concentration of ~10 uL/0.5 mL.
This concentration of DMSO did not have an observable effect on the bacteria when examined microscopically. Stock solutions of 10 mM TRIS/3.3 mM citric acid were prepared and diluted 10 times for a working solution concentration of 1 mM TRIS/0.33 mM citric acid as needed. Surfactants were added to these working concentration buffers in the appropriate concentrations as they were needed daily. Sodium hydroxide and hydrochloric acid were used to adjust the pH when necessary. The standard running buffer concentration is 1 mM TRIS/0.33 mM citric acid with 1 mg/mL CTAB, pH 7, unless otherwise noted in the figure legends. The standard sample buffer is 1 mM TRIS/0.33 mM citric acid, pH 7.
The CE separations were performed on a Beckman Coulter P/ACE MDQ capillary eletrophoresis system equipped with a 488 nm laser-induced fluorescence and a photodiode array detector. UV detection was used in all experiments except those that involved the use of S. aureus. Laser-induced fluorescence detection was used in these instances since it provided a more intense signal. The result can be illustrated as Figure 3, where the microbial band migrates later than the EOF front and is detected as a sharp peak. Other molecular contaminants are eluted before the EOF peak, with the peak, between the EOF and Microbial peak, or on occasion, after the microbial peak. The microbe peak is free of neutral entities, which travel with EOF, and any other non- microbial material.
Example 2: Single-cell Detection
Laser-induced fluorescence may theoretically be able to detect extremely low levels of bacteria. This example describes the method for single-cell detection by using fluorescent microbe dyes. To experimentally determine that a sample contains no microbial cells (a sterile sample), a detection limit of a single cell must obtained. Suitable fluorescent dyes are the dyes with the absorption and emission wavelength away from the common background absorption or emission. Examples of these dyes include Baclight fluorescent dyes.
Materials. Buffer additives, including tris(hydroxymethyl)aminomethane (TRIS), cetyltrimethylammonium bromide (CTAB), sodium hydroxide, and hydrochloric acid were obtained from Aldrich Chemical (Milwaukee, WI). Citric acid was purchased from Fisher Scientific (Fair Lawn, NJ). Caprylyl sulfobetaine (SB3-10) was ordered form Sigma (St. Louis, MO). Molecular Probes, Inc. (Eugene, OR) supplied the BacLight Green fluorescent dye. Nutrient and brain heart infusion broths were products of Difco Laboratories (Franklin Lakes, NJ), while luria broth was obtained from Sigma. Brevibacterium tapei (ATCC no. 13744), Corynebacterium acetoacidophilum (ATCC no. 13870), Escherichia blattae (ATCC no. 29907), Bacillus cereus (ATCC no. 10702), Bacillus subtilis (ATCC no.12695), Bacillus megaterium (ATCC no. 10778), Candida albicinas (ATCC no. 10231), and Rhodotorula (ATCC no. 20254) were all purchased from American Type Culture Collection (Manassas, VA). Uncoated fused silica capillaries with inner and outer diameters of 100 μm and 365 μm respectively, were purchased from Polymicro Technologies (Phoenix, AZ). All microorganisms examined
in this study are rated biosafety level one. Therefore, standard microbiological practices may be employed.
Methods. Analyses were performed on a Beckman Coulter P/ACE MDQ capillary electrophoresis (CE) system equipped with a photodiode array and a 488 run laser-induced fluorescence (LIF) detector (Fulleiton, CA). Capillaries used were 30 cm in total length (20 cm to the detector). New capillaries were initially conditioned with the following rinses: 1 N NaOH, 1 N deionized water, 1 N HCl, and running buffer each for 3 min. Between runs, the capillaries were washed with 1 N NaOH, deionized water, and running buffer for 1 min each. Working buffers were prepared by adding appropriate amounts of TRIS and citric acid to deionized water to produce a 10 mM TRIS/3.3 mM citric acid solution, and diluting this solution 1 Ox to a final concentration of 1 mM TRIS/ 0.33 mM citric acid. pH was adjusted to 7 using dilute sodium hydroxide or hydrochloric acid. The final running buffers were prepared by dissolving CTAB in the working buffer to a concentration of 1 mg/niL. Blocking solutions contained SB3-10 of varying concentrations in working buffer. These solutions were all made fresh daily. All bacteria and fungi were grown as specified by the supplier. Initially, the microorganisms were grown in the appropriate liquid broth, and then plated on agar growth media and stored under refrigeration. All broths and agar were autoclaved (Primus autoclave, Omaha, NE) for 1 hr prior to inoculation. For experiments, fresh liquid broth was inoculated with a single microbe colony that was extracted from the agar plate. These cells were grown at 30-37 0 C under gentle agitation for approximately 24 hrs, producing a cellular concentration of ~10 CFU/mL. The microorganisms were centrifuged down, and the excess broth was removed. The cells were then washed with working TRIS/citric acid
buffer, recentrifuged, and finally resuspended in fresh buffer for analysis. BacLight Green fluorescent dye was used to stain the cells for LIF detection. This dye was initially prepared in DMSO to produce a 1 mM solution, as directed by the manufacturer. The cells were stained by adding 1 μL of dye solution per 1 rnL of microbial solution and incubating the cells at room temperature for at least 15 min.
After all wash cycles, the capillary was filled with running buffer containing CTAB. Three injections were made prior to the run: 1) sample plug consisting of microorganisms, 2) spacer plug of running buffer, 3) blocker plug of SB3-10. Unless otherwise noted, sample injections were made for 5 s at 0.5 psi, spacer injections for 4 s at 0.5 psi, and blocker injections for 2 s at 0.1 psi. For single cell analyses, microbial solutions were diluted down to ~10 4 CFU/mL and stained with BacLight Green dye as described above. A small drop (~2 uL) of this solution was applied to a sterile microscope slide, and using an autoclaved micro-utensil the drop was smeared across the slide to produce numerous drops of smaller volume. These drops were then inspected visually by microscopy until a drop that contained only a single microorganism was identified and isolated. This entire drop was then injected into the capillary via capillary action (in place of the sample plug mentioned above). All run buffers, solutions, and vials used in the CE analysis were autoclaved prior to the run. Run voltage was set to -2 kV in reverse polarity (current: -1.4 μA), due to reversal of the electroosmotic flow (EOF) by CTAB.
In the above examples, similar electropherogram as Figure 3 may be obtained when the UV detector is set to 449 run. The bacteria in the sample injection are the only species present capable of giving a signal (largely due to Mie scattering) when detected at
449 nm, hence the resulting sharp peak in the electropherogram shows only the presence of those microbes.