Patent Application: US-30062807-A

Abstract:
the invention provides compositions and methods for determine the number of bacteria and other microbes in samples having low concentrations of microbes , for use , e . g ., in biological warfare defense , microbe detection and agricultural and environmental sciences .

Description:
the invention provides compositions and methods for the enumeration of microbial cells without culturing . the compositions and methods of the invention can be used for microbial ecology and / or water quality evaluation . the invention utilizes bulk fluorescence using the sybergold ™ ( invitrogen - molecular probes , eugene oreg .) dna stain , or equivalent , to rapidly estimate microbial numbers in an environmental sample , a soil or water environment , including , e . g ., a fresh , marine , and / or estuarine water environmental sample . the bulk fluorescence method of the invention is comparable to estimating cell concentrations in cultures using optical density . the enhanced method of the invention enables the user to estimate microbial numbers at low concentration , e . g ., the concentrations found in an environmental sample . the methods of the invention work in both single - cell and 96 - well plate fluorescent spectrophotometers . in practicing the invention , differences of approximately 10 5 cells per ml were discernable and the precision of the bulk fluorescence was higher than direct counting by epifluorescent microscopy . in one aspect , treatment with dnase i is used , it increases sensitivity by lowering background noise attributed to free dna . this embodiment of the invention is a simple , rapid , inexpensive technique which is adaptable for automatically estimating microbial numbers in samples . in practicing the invention , amplification reactions can be used to quantify the amount of nucleic acid in a sample , label the nucleic acid ( e . g ., to apply it to an array or a blot ), detect the nucleic acid , or quantify the amount of a specific nucleic acid in a sample . in one aspect of the invention , message isolated from a cell or a cdna library are amplified . the skilled artisan can select and design suitable oligonucleotide amplification primers . amplification methods are also well known in the art , and include , e . g ., polymerase chain reaction , pcr ( see , e . g ., pcr protocols , a guide to methods and applications , ed . innis , academic press , n . y . ( 1990 ) and pcr strategies ( 1995 ), ed . innis , academic press , inc ., n . y ., ligase chain reaction ( lcr ) ( see , e . g ., wu ( 1989 ) genomics 4 : 560 ; landegren ( 1988 ) science 241 : 1077 ; barringer ( 1990 ) gene 89 : 117 ); transcription amplification ( see , e . g ., kwoh ( 1989 ) proc . natl . acad . sci . usa 86 : 1173 ); and , self - sustained sequence replication ( see , e . g ., guatelli ( 1990 ) proc . natl . acad . sci . usa 87 : 1874 ); q beta replicase amplification ( see , e . g ., smith ( 1997 ) j . clin . microbiol . 35 : 1477 - 1491 ), automated q - beta replicase amplification assay ( see , e . g ., burg ( 1996 ) mol . cell . probes 10 : 257 - 271 ) and other rna polymerase mediated techniques ( e . g ., nasba , cangene , mississauga , ontario ); see also berger ( 1987 ) methods enzymol . 152 : 307 - 316 ; sambrook ; ausubel ; u . s . pat . nos . 4 , 683 , 195 and 4 , 683 , 202 ; and sooknanan ( 1995 ) biotechnology 13 : 563 - 564 . the invention will be further described with reference to the invention described in the following appendix ; however , it is to be understood that the invention is not limited to such examples . while the invention is susceptible to various modifications and alternative forms , specific examples thereof have been shown by way of example in the drawings and are herein described in detail . it should be understood , however , that the invention is not to be limited to the particular forms or methods disclosed , but to the contrary , the invention is to cover all modifications , equivalents , and alternatives falling within the spirit and scope of the claims . enumeration of microbial cells without culturing is an essential technique for microbial ecology and water quality evaluation . here we show that bulk fluorescence using the sybr gold dna stain can be used to rapidly estimate microbial numbers in fresh , marine , and estuarine waters . the bulk fluorescence method is comparable to estimating cell concentrations in cultures using optical density , however , this enhanced method enables the user to estimate microbial numbers at lower concentration and found in environmental samples . the technique worked in both single - cell and 96 - well plate fluorescent spectrophotometers . differences of ˜ 10 5 cells per ml were discernable and the precision of the bulk fluorescence was higher than direct counting by epifluorescent microscopy . treatment with dnase i increased sensitivity by lowering background noise attributed to free dna . this technique is simple , rapid , inexpensive , and adaptable for automatically estimating microbial numbers in water samples . a major breakthrough in microbial ecology was direct counting of microbial cells via dapi staining and epifluorescence microscopy ( hobbie et al ., 1977 ). using this method , it was established that there are ˜ 10 6 prokaryotic cells per milliliter of seawater and ˜ 10 9 prokaryotic cells per gram of soil or sediment , despite the fact that only 1 % of these cells are readily culturable . direct count protocols have been modified to incorporate newer nucleic acid stains , such as sybr green ( noble and fuhrman , 1998 ) and sybr gold ( breitbart et al ., 2004 ). the sybr dyes have a greater fluorescence enhancement ( i . e ., increase in fluorescence when the dye binds to dna ) than ethidium bromide or dapi . sybr nucleic acid stains also have a higher fluorescence quantum yield , making them more sensitive . these characteristics have made the direct counts of prokaryotic cells , and even viruses , relatively routine ( noble and fuhrman , 1998 ). further improvements have included the adaptation of flow cytometry and automated image analyses software . despite these improvements , current methods for counting microbes are relatively slow and require expensive / sophisticated equipment ( e . g ., flow cytometers or epifluorescent microscopes ). an inexpensive , rapid technique for estimating microbial concentrations would be valuable to investigators in several fields including water quality assessment , aquaculture , and microbial ecology . here , we present a rapid method for estimating microbial numbers in marine , estuarine , and fresh water samples using simple fluorometry and the nucleic acid stain sybr gold . correlation of microbial numbers to sybr gold bulk fluorescence : to determine if bulk fluorescence could be used to quantify microbes , a dilution series was created using varying proportions of 0 . 45 μm (+ microbes ) and 0 . 02 μm filtered seawater (− microbes ). the different dilutions were stained with sybr gold and the relative fluorescence units ( rfus ) measured on a hitachi f4500 fluorometer . as shown in fig1 a , there was a correlation between the dilutions and bulk sybr gold fluorescence . there was also a strong correlation ( r 2 = 0 . 94 ) between the relative fluorescence as measured by the fluorometer and the number of microbes in the samples , which were counted manually using epifluorescence microscopy ( fig1 b ). a similar dilution series was also measured on the gemini xs 96 - well fluorometer . there were significant differences ( p & lt ; 0 . 05 ) between 0 . 45 μm samples diluted by 40 % with 0 . 02 μm filtered seawater ( e . g ., 20 % vs 60 % and 60 % vs 100 % in fig1 c ). the r 2 value for the seawater dilution series measured on the gemini fluorometer was 0 . 86 ( fig1 d ). these preliminary analyses suggested that bulk fluorescence with sybr gold may be used to enumerate microbes on both single - cell and multi - well fluorometers . microbial numbers in environmental water samples : bulk fluorescence and direct counts were performed on fresh , marine , and estuarine water samples collected from the san diego area . as shown in fig2 , there were visibly discernable differences between samples that vary by at least 5 × 10 5 cells ml − 1 . precision : the precision of the bulk fluorescence method versus direct counts was compared in three water samples . the error between samples was estimated using the standard deviation of the mean from the three replicate samples measured per site . the standard deviation in rfus for the three samples ranged from 12 to 18 ( fig3 ). estimates of total bacterial numbers using epifluorescent microscopy ranged from 9 . 1 × 10 5 to 1 . 0 × 10 6 and the standard deviation ranged from 7 . 2 × 10 4 to 1 . 3 × 10 5 ( fig3 ). in the south san diego bay water sample , error with the fluorometer was 2 . 8 % and error with manual counting was 12 %; in the harbor island sample , error with fluorometer was 2 . 9 % and error with manual counting was 15 %; and in the shelter island sample , error with fluorometer was 4 . 2 % and error with manual counting was 7 . 1 %. therefore , the precision was higher with the fluorometer then with manually counting using epifluorescent microscopy . treatment with dnase i lowers background and increases sensitivity : dissolved dna has been found at concentrations as high as 5 - 44 μgl − 1 in estuaries and 2 - 15 μgl − 1 for coastal oceanic environments ( deflaun et al ., 1987 ; paul et al ., 1987 ; boehme et al ., 1993 ; jiang and paul , 1995 ). therefore , it is likely that dissolved dna will cause background noise in the bulk fluorescence signal . to test this , 5 water samples were treated with dnase i and assayed with the bulk fluorescence protocol . the addition of the dnase i lowered background noise associated with the blank ( fig4 ). for example , the signal in the anthony &# 39 ; s pier , laurel st ., and south fiesta island samples was completely masked by the free dna noise . therefore , we recommend that a dnase i step be included when using this method . the sybr gold and dnase i can be added simultaneously ( data not shown ). the effects of preservatives : to determine if the bulk fluorescence method was compatible with common preservation techniques , 3 different samples were harvested and stored at 4 ° c . or ˜ 20 ° c . or treated with 2 % paraformaldehyde or 0 . 1 % sodium azide . as shown in fig5 , the differences in the rfus for the samples and blanks were not consistent for any of the preservation techniques over time . paraformaldehyde fixing seemed to be the best , but even those samples show variation over time . other research has shown that microbial numbers , as determined by direct counts , can change over time even after the addition of preservatives ( wen et al ., 2004 ). therefore , samples should be analyzed as soon as possible in order to obtain the most accurate reading . the effects of high versus low dna / rna content in cells : vibrio parahemalyticus was used to determine if there was a significant difference in the fluorescence of actively growing cells versus dormant cells . a high concentration sample (× 10 7 cells ml 1 ) and a low concentration sample (× 10 6 cells ml − 1 ) were both used for the comparison . stationary cells had rfus that were ˜ 44 % lower than those cells in log phase . presumably , the extra fluorescence was due to higher rna content . this implies that bulk fluorescence will be higher for actively growing microbial communities . in natural environments , ˜ 50 % of the microbes are categorized as high dna / rna content cells ( gasol et al ., 1999 ). therefore , in most environmental samples , the high and low dna / rna content cells would balance . recommended protocol : based on the results presented above , we suggest the following protocol . syringe filter ( 0 . 45 μm for sample and 0 . 02 μm for blank ) 1 ml of water sample into an eppendorf tube containing λ1 of 10 , 000 × sybr gold and 13 units ml − 1 of dnase i . incubate for one minute . measure the emission spectrum 450 - 650λ with an excitation of 495λ . calculate area under the curve , σ ([ f ( x n ) δx − f ( x n - 1 ) δx ]/ 2 ). if a plate reading fluorometer is available , replicates should be measured and averages calculated . to calibrate the curve for a particular water type , initial direct counts with epifluorescent microscopy should be performed . measurement of environmental samples using recommended protocol : water samples were collected from freshwater and seawater in order to test the recommended protocol . both freshwater and seawater fluorescent measurements showed a strong correlation , r 2 = 0 . 96 and 0 . 92 respectively , when compared to direct counts ( table 1 ). it can be concluded from these measurements that this bulk fluorescence method may be useful in estimating microbial abundances in both freshwater and seawater . approximation of microbial concentrations without direct counts : table 2 represents a guide for estimating cell concentrations using relative measurements of similar water samples . this is only a guide for rapid approximations . investigators should construct similar tables for their environment of choice . potential for automation : an automated version of this protocol would need to include 0 . 45 and 0 . 02 μm filtration , an injection unit for mixing the sybr gold and dnase i , a fluorometer , and a data recorder . miniature fluorometers are commercially available ( e . g ., ocean optics ; dunedin , fla .) and custom - built basic fluorometers are inexpensive to build . such systems would be very good at estimating relative microbial concentrations . for example , most of the remote buoy systems for measuring total chlorophyll could be easily adapted to estimating microbial numbers using the bulk fluorescent method . simple photomuliplier tube - uv systems would be particularly useful for routine monitoring of aquaculture ponds or storm drains in a city . we need methods for remote sensing of microbial dynamics . bulk fluorescence of sybr gold , as presented here , is a reasonable candidate for estimating numbers because it is robust and it can be performed with very simple , cheap equipment . water sampling : all water samples were collected from the san diego area , including mission bay , san diego bay , la jolla shores , imperial beach ( tb ), ocean beach ( ob ), ob estuary , torrey pines , lake lindo , lake murray , the old mission dam and the san diego river . portions of each sample were put through a 0 . 45 μm or 0 . 02 μm pore syringe filter . the 0 . 45 μm filter removes protists and eukaryotic algae and was used as the sample . the 0 . 02 μm filter removes the microbial cells and viral particles and was used as the blank . staining with sybr gold and bulk fluorescent measurements : both a hitachi f4500 ( schaumburg , ill .) and a gemini xs ( molecular devices corporation ; sunnyvale , calif .) were used in these experiments . water samples measured using the single - cell hitachi fluorometer were prepared by mixing 1 ml of the sample or the blank with 1 μl 10 , 000 × sybr gold nucleic acid stain ( molecular probes ; eugene , oreg .). the relative fluorescence was measured using an excitation wavelength of 495λ and an emission spectra ranging from 450 - 650λ . for measurements using the gemini fluorometer 1 μl of 10 , 000 × sybr gold was added to 1 ml of sample or blank , then 200 μl aliquots were pipetted into 4 wells on a 96 - well plate . the parameters set for the excitation and emissions wavelengths on the gemini fluorometer were identical to those set on the hitachi , except , a cut - off filter at 515λ was used . a cutoff filter was not available on the hitachi , nevertheless , the emission curves generated by both fluorometers were similar . epifluorescence microscopy : cells were counted by filtering samples fixed in 2 % paraformaldehyde onto a 0 . 02 μm anodisc ( whatman ), staining with sybr gold ( molecular probes , inc ), and directly counting by epifluorescent microscopy . cells were visualized at 1000 × using a leica dm rbe microscope equipped for epifluorescence with a mercury bulb ( 100 w ) and filter set xf57 - 1 ( omega ). images were captured using a ccd camera ( olympus america ) and cells were counted (& gt ; 200 per sample ) in 10 - 20 fields selected at random . seawater dilution series : one liter of seawater was collected from the san diego bay . the seawater was 0 . 45 μm filtered and then diluted by 1 / 5 , 2 / 5 , 3 / 5 , and 4 / 5 using 0 . 02 μm filtered seawater collected from the same site . one μl of 10 , 000 × sybr gold dna stain was added to 1 ml of each dilution . the whole seawater and the 4 seawater dilutions were measured for their total fluorescence using both the hitachi f4500 fluorescence spectrophotometer and the gemini xs fluorometer . the area of each scan , σ ([ f ( x n ) δx − f ( x n - 1 ) δx ]/ 2 ), was calculated for the diluted samples and the blank ( 1 ml of 0 . 02 μm filtered seawater ). for the dilution series measured using the gemini fluorometer , two - tailed t - tests were used to determine whether significant difference existed between the averages obtained from the four replicates measured per sample and the percentage of bacteria in the sample . three ml of each dilution were also fixed in 2 % paraformaldehyde and the microbes (& gt ; 200 cells ) were counted using epifluorescent microscopy . field tests : fifty ml water samples were collected from 6 different sites in san diego , calif . samples were collected from fiesta island , san diego bay , mission bay , point loma , ocean beach estuary , and the san diego river . each water sample was 0 . 45 μm syringe filtered to remove large particles and eukaryotic organisms . one μl of 10 , 000 × sybr gold dna stain was added to 1 ml of each sample and measured for its total fluorescence using a hitachi f4500 fluorescent spectrophotometer . the emission spectrum of 450 - 650λ was collected ( excitation = 495λ ). after a water sample was scanned , it was immediately pipetted out of the cuvette , and fixed with 2 % paraformaldehyde for the direct counts . the area of each wavelength scan was calculated for all water samples . precision : to measure the precision of the fluorescence spectrophotometer , water samples were collected from the shipyard at san diego bay , harbor island , and shelter island . all three samples were 0 . 45 μm syringe filtered to remove large particles and eukaryotic organisms . one μl of 10 , 000 × sybr gold was added to 1 ml of each seawater sample . the three different water samples were measured 3 separate times for their total fluorescence using the hitachi f4500 and counted three separate times using epifluorescent microscopy . dnase i treatments : two μl of 6475 units ml − 1 dnase i ( sigma ; st louis , mo .) was added to 1 ml of water sample and incubated for 5 minutes at room temperatures . samples for this experiment were measured on the gemini fluorometer . the relative fluorescence was estimated by calculating the area under the emission spectrum . four replicates were measured per sample and averages were calculated . effects of preservatives : to test the effects of different preservatives on the sybr gold bulk fluorescence method , water samples collected from 3 different sites were stored at 4 ° c ., − 20 ° c ., treated with 2 % paraformaldehyde , or 0 . 1 % sodium azide . fluorescence was measured at 0 , 24 , 48 hrs , and 1 week post - preservation using the gemini fluorometer . the relative fluorescence was estimated by calculating the area under the emission spectrum . four replicates were measured per sample and averages were calculated . quantity of relative fluorescence in actively growing versus dormant cells : vibrio parahemalyticus was used as a model to test the effects of cells with high dna / rna content versus low dna / rna content on our bulk fluorescence methods . an overnight culture of v . parahemalyticus was grown at 37 ° c . in lb broth . three different dilutions of overnight were added to three flasks containing 250 ml of 0 . 2 μm filtered autoclaved seawater . cells were grown at room temperature with aeration for 12 hours ( high dna / rna content cells ) and for 72 hours ( low dna / rna content cells ). sub - samples were taken , dilutions were made , and cells were measured for their fluorescence on the gemini fluorometer using the methods previously described . one ml of each sample dilution was fixed in 2 % paraformaldehyde and counted using epifluorescent microscopy . preparation and measurement of samples using “ recommended ” conditions : four freshwater and seven seawater samples were collected from the san diego area . water samples were 0 . 45 μm filtered ( sample ) and 0 . 02 μm filtered ( blank ) as previously described . two μl of 6475 units ml − 1 dnase i was added to one ml of both filter fractions per sample and incubated for 5 minutes . one μl of 10 , 000 × sybr gold was added and water samples were measured using the gemini fluorometer as previously described . the relative fluorescent units ( rfu ) were generated by calculating the area under the emission spectrum and subtracting the background fluorescence generated from the blank measurement . one ml of each water sample was fixed in 2 % paraformaldehyde and counted using epifluorescent microscopy this research was supported by the spawar systems center san diego independent research ( ir ) program and the national science foundation aquatic phage grant ( nsf03 - 16518 ). the authors thank mya breitbart for her contributions to the project , beltran rodriguez - brito for his expertise on the mathematical and statistical calculations , and steve barlow at the sdsu - emf for help with the microscopy . seawater samples were collected from 4 open ocean locations in imperial beach ( ib ) and coronado island , as well as 3 bay locations ( tijuana slough , ocean beach ( ob ), and mission beach ( mb )). relative fluorescent units ( rfu ) after background substitution calculations were generated by subtracting the sample measurement from the blank measurement . correlation between rfu and manual counts represents the r 2 value generated from the four freshwater samples and from the seven seawater samples . fig1 . measurement of bulk fluorescence in seawater dilution series using the hitachi and gemini xs fluorometers . the relative fluorescence was measured on the hitachi fluorescent spectrophotometer using an excitation wavelength of 495λ and an emission spectra ranging from 450 - 650λ . the area under the curve was calculated and a xy scatter plot was constructed ( a ). correlation between bulk fluorescence and manual cell counts in the seawater dilution series ( b ). measurement of relative fluorescence in a seawater dilution series using gemini fluorometer ( c ). the bulk fluorescence was estimated by calculating the area under the emission spectrum and a bar graph was constructed . four replicates per sample were measured and averages were calculated . correlation between relative fluorescence and percentage of whole seawater in sample ( d ). the gemini fluorometer had the capability of measuring several replicates simultaneously , while the hitachi could only measure single - cells . fig2 . measurement of bulk fluorescence in six san diego water samples . samples were collected from mission bay ( mb ), ocean beach ( ob ) estuary , point loma , san diego ( sd ) bay , and the san diego river . each water sample was measured for the total fluorescence using a hitachi f4500 fluorescent spectrophotometer . the emission spectrum of 450λ - 650λ was collected ( excitation = 495λ ). the area of each wavelength scan was calculated for all water samples . after a water sample was scanned , it was immediately pipetted out of the cuvettes and fixed with 2 % paraformaldehyde for the direct counts by microscopy . fig3 . precision of the measurements for relative fluorescence in three seawater samples using hitachi fluorescent spectrophotometer . the standard deviation in rfus for the three samples ranged from 12 to 18 . estimates of total microbial numbers using epifluorescent microscopy ranged from 9 . 1 × 10 5 to 1 . 0 × 10 6 and the standard deviation ranged from 7 . 2 × 10 4 to 1 . 3 × 10 5 . the average error using the fluorometer was 3 . 3 %, while the average error for manual counts was 11 %. fig4 . the effects of dnase i on the relative fluorescence of seawater samples . five water samples were treated with dnase i and compared to untreated samples with the bulk fluorescence protocol . the 0 . 02 and 0 . 45 μm filtered curves represent averages of four replicates . fig5 . the effects of preservatives on seawater samples . three seawater samples were stored at 4 ° c ., − 20 ° c ., treated with 2 % paraformaldehyde , or 0 . 1 % sodium azide . fluorescence was measured at 0 , 24 , 48 hrs , and 1 week post - 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