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reo SCIENCE I SPECTRUM Jo 1216 STATE STREET • POST OFFICE BOX 3003 • SANTA BARBARA, CALIFORNIA 93105 TELEPHONE (805) 963-8605 A NEW TECHNIQUE FOR STUDYING MICROORGANISMS With differential light scattering you can learn more in minutes about microbial morphology and physiology than others have learned in years. Some examples are given in the accompanying Application Notes. This technique - new to the study of microorganisms - shares some capabilities of optical and electron microscopy. Sample preparation is simple and rapid; submicron physical details can be studied; a wide variety of samples can be examined in many different environments and states, and physical changes monitored. The differential light scattering measurements detailed in the accompanying Application Notes were made with our DIFFERENTIAL I photometer. In addition to determining structural such measurements enable parameters~ bacterial presence and concentration to be determined easily. The scattering data can be analyzed easily and quite accurately using rapidly~ the Atlas of Light Scattering Curves described in the enclosed flyer. For more information on these or other or on our products, applications~ simply fill out the enclosed prepaid card. A copy of our current seminar schedule also is enclosed. If you have any question regarding the study or results reported in the Application Notes, please let us know. Sincerely yours, SCIENCE SPECTRUM, INC. 92~ James E. Hawes Vice President, Marketing Enclosures 'lpproved for Releas~ ,Pate 2 7 Ff:R 1Ci~ //reo ,, ,. ~sciENCE I SPECTRUM Jo 1216 STATE STREET • POST OFFICE BOX 3003• SANTA BARBARA, CALIFORNIA 931C TELEPHON (aos) 963-asc SEMINARS Seminars with demonstrations explaining light scattering theory and its many applications are offered by Science Spectrum periodically at various central locations throughout the United States, free of charge. The DIFFERENTIAL I and DIFFERENTIAL II instruments are also exhibited at selected professional meetings. The seminars and exhibits currently scheduled are: September 14- 16~ 1971 Washington~ D. C. - Exhibit September 17, 1971 Washington. . D. C. - Seminar September 21 .. 1971 Raleigh, North Carolina - Seminar September 24 .. 1971 New York. . New York - Seminar September 27 .. 1871 Boston.. Massachusetts - Seminar \ ·' October 5 .. 1971 Los Angeles.. California - Seminar October 7.. 1971 San Francisco, Calif. - Seminar (tentative) October 15.. 1 971 Chicago, Illinois - Seminar (tentative) If you wish to attend one of the seminars listed above, please complete the form below and return it to the The image displays a digital representation of a document's header or introductory page. On the left, there is a stylized graphic of a vault door, suggesting themes of secrecy and access to information. To the right of the vault, large, stylized white text reads "THE BLACK VAULT," serving as the title. Below this title, a block of text explains that the document was obtained from "The Black Vault," an online database of declassified government documents, specifically noting its inclusion in the MKULTRA/Mind Control Collection, a compilation of over 20,000 pages declassified by the CIA. The text concludes with a URL for accessing the collection: http://mkultra.theblackvault.com. No photographs, handwritten annotations, official stamps, forms, diagrams, tables, or redactions are visible. \ ·' October 5 .. 1971 Los Angeles.. California - Seminar October 7.. 1971 San Francisco, Calif. - Seminar (tentative) October 15.. 1 971 Chicago, Illinois - Seminar (tentative) If you wish to attend one of the seminars listed above, please complete the form below and return it to the Company., A program will be sent to you about two weeks before the scheduled meeting. . together with a confirmation of your reservation. I will attend your seminar to be held on ------------------------------------------- at (city) Please send my prograrr ----------------------------------------------- to: NAME - ------------------------------------ TITLE- ----------------------- INSTITUTION- -------------------------------------------------------- STREET ------------------------------------------------------------------ CITY, STATE, ZIP ------------------------------------------------------- TELEPHONE- -( -------------------------------------EXTENSION- --------- Atlas of Light Scattering Curves. l.Sf Introduction VERTICAL The interpretation of light scattering data has R = 500 nm long been an obstacle to the widespread use of this powerful analytical tool. While some scientists de voted their careers to the theoretical understanding 8,; of light scattering, their results were not readily adapt 1.39 ed for use by workers in other fields. High speed digital computers can now be used economically to 1.33 generate scattering data for a variety of model particles. "' The purpose of the Science Spectrum Light Scattering Atlas is to make this computer-generated information available in a convenient form for a wide range of light scattering applications involving small particles. Computer-generated Ii ght scattering patterns are plotted on the same scale as the experimental data measured by the Differential/ and II light scat tering photometers. Semitransparent vellum paper has A sample page from the Atlas showing the light scattering been used for the Atlas so that accurate comparison curves for homogeneous spherical particles of 500 nm radius and four different refractive indices. of theory and experiment is achieved by merely over laying the two sheets. Tables of normalization con the ~cattering curves of spheres with radius between stants for absolute scattering power are also provided. 0.05 and 1 micrometer in steps of 0.05 micrometer, for refractive indices ranging from 1.33 to 1.59. The Single Spherical Particles in Air refractive index of the surrounding medium is that of air (n - 1.0). Curves for both linear polarizations A wide variety of processes produce homogen :?ous spherical particles of approximately one microm are given. Inspection of the scattering atlas for spheri ~ter in diameter. For example, photochemical aerosol cal particles shows that particle size can easily be x "smog" droplets The document is a single-page letter from Science Spectrum, Inc. addressed to an unknown recipient. It features the company's logo and contact information at the top, with a title below indicating it's about a "New Technique for Studying Microorganisms." The body of the letter is text-based, describing a method of microbial analysis. At the bottom, there is a handwritten signature "Jh Hawa" and printed name "James E. Hawes," followed by his title. Below this, "Enclosures" is noted. In the lower right corner, there is a stamp indicating "Approved for Release," with a date "27 FEB 1979," and a circled handwritten number "130." (n - 1.0). Curves for both linear polarizations A wide variety of processes produce homogen :?ous spherical particles of approximately one microm are given. Inspection of the scattering atlas for spheri ~ter in diameter. For example, photochemical aerosol cal particles shows that particle size can easily be x "smog" droplets and colloidal particles like those specified to within 0.1 micrometer diameter. A de n latex paints are spherical. Such small particles may termination of refractive index to well within ten per :asily be suspended in air by nebulizing a liquid sus cent accuracy is achieved for spheres by simply exam )ension and their individual scattering patterns are ining the relative intensities at peak amplitudes. eadily measured with the Differential// scattering Supplements Jhotometer. Periodically, additional scattering curves are pub This important class of scattering objects is com lished as supplement sections to the Atlas. Owners of letely described by two parameters: radius and re the Science Spectrum Scattering Atlas will receive all ·active index. The first section of the Atlas displays supplements issued within two years of the date of purchase without charge. Subjects selected for early diameter at a constant refractive index can be \.. supplements include: the effect of size distribution vided for studies of colloidal size distributions. B\ upon scattering from suspensions of spheres; the effect this means sizing accuracy of± 10 nm diameter can upon scattering of size and size distribution changes be obtained as reported by Phillips eta/ in the J. in model bacteria and mitochondria; scattering from Colloid Int. Sci. 34 (1970), p. 159. Scattering curves conductive particles; scattering from absorbing parti· on absorbing spheres can also be computed as needed. cles; and scattering from airborne bacteria. Measured Curves on .any spherically symmetric structure with scattering curves from known non-spherical particles varying complex refractive index can be generated. may also be provided. Specific applications for specialized shell structures include bacteria, bacterial spores, microencapsula· Computation of special scattering curve sets for tion particles, compound aerosol particles with large a wide variety of objects will be done at moderate nuclei, etc. Even more varied shapes can be computed cost using proprietary Science Spectrum computer exactly when the particle nearly matches the refrac codes. The scattering from spheres of different diam· tive index of the medium in which it is immersed - eters or refractive index can be computed on order. as in the case of bacteria in The page displays a formal document with an official header featuring the "SCIENCE SPECTRUM" logo. It outlines a schedule of seminars and their locations across the United States. Below the seminar schedule is a form for attendees to fill out, requesting their name, institution, street address, city, state, zip code, and telephone number. The form also includes fields for "Please send my program" and "TITLE," as well as "EXTENSION," indicating a request for supplementary materials or information related to the seminar. There are no photographs, handwritten annotations, official stamps, diagrams, tables, or visible evidence of experimental procedures on this page. computed cost using proprietary Science Spectrum computer exactly when the particle nearly matches the refrac codes. The scattering from spheres of different diam· tive index of the medium in which it is immersed - eters or refractive index can be computed on order. as in the case of bacteria in water. For example, curves for small variations in particle Please send me ____c opies* of the Science Spectrum Scattering Atlas at $25.00 each, plus $1.25 sales tax if delivered in California. I understand that I will receive, without further charge, all supplemental sections to the Atlas published in the next two years. Also, if payment is enclosed with my order, Science Spectrum will pay shipping costs. If I am not satisfied with the Atlas, I may return it postpaid within 10 days for a full refund. Name _______________________________________________________________ __ Title ___________________________________ Department_ ______________________________________________________________ Organization ________________________________________________ Street._ ___________________________________________________________ City, State, and Zip ______________________________ My main interest in light scattering is: _______________________________________ The supplement I am most interested in is: _________________________________________ * Purchasers of a Differential/ or Differential// photometer receive with the instrument two copies of the Atlas and all supplements for two years. AI-17-QSI (2) Physiological Monitoring of Bacteria and Mitochondria Introduction servatives such as phenol, formalin, or alcohols can be measured precisely4. Subtle changes in response to elevated Optical methods ranging from microscopy to turbidi· temperatures5 or pressures are easily determined. Size metry have long been used to monitor bacterial growth and modification and cellular damage occuring in phage-infected division. However, the optical microscope is unable to re· bacteria can be measured. The process of spore germination solve features smaller than a few wavelengths of light in size. can be monitored as it proceeds. The response of chloro· Turbidimetric measurements are subject to large errors plasts to various processes including photophosphorylation because the attenuation of light is a function of the product have been followed by light scattering6·8. The susceptibility of particle scattering cross section and particle density. of bacteria to various antibiotics can be measured with1n Since the particle scattering cross section is not in general minutes of contact9. Changes in mean cell size during syn· the same as the particle's geometrical cross section, signifi· chronous growth can be monitored with an accuracy of cant interpretive problems arise. A given value of transmit· ± 20 nm. The effect of different growth media on the size tance will often correspond The document page features a scientific graph displaying "light scattering curves" for spherical particles, with axes labeled "SCATTERING ANGLE" and "RELATIVE INTENSITY." The graph shows multiple wavy lines indicating variations in intensity at different angles. Above the graph, there is a title "ATLAS" and information about "VERTICAL," "R = 500 nm," and a copyright notice. The left side of the page contains text under the headings "Introduction" and "Single Spherical Particles in Air." There are no photographs, handwritten annotations, stamps, forms, or redactions visible. The visual content is primarily composed of text and a scientific illustration. Changes in mean cell size during syn· the same as the particle's geometrical cross section, signifi· chronous growth can be monitored with an accuracy of cant interpretive problems arise. A given value of transmit· ± 20 nm. The effect of different growth media on the size tance will often correspond to several different products of distribution of cells can be seen clearly via light scattering particle density and particle size. On the other hand, differ· patterns. These measurements can be made without dis· entia! light scattering measurements (i.e., recording the turbing the growth of the culture. Some details of such ) pattern of light scattered by such particles as a function of studies are discussed below. angle relative to the direction of the illuminating beam) are unambiguous, often yielding size and shape information of much higher precision than obtainable with a microscope. LASER CUVETTE t Under optimum conditions, cell size determinations of± 2% accuracy are achievable with differential light scattering 0~ )lro measurements. >::> Figure 2 DETECTOR The Differential I Photometer The Differential I light scattering photometer is a highly versatile instrument, uniquely suited to the study of liquid suspensions of bacterial cells. It is shown in Fig. 1 and its operation is represented schematically in Fig. 2. In use, a cuvette containing the suspension is placed in the instru· ment and illuminated by the intense monochromatic beam Figure 1 of an argon-ion laser. A specially designed scanning detector e system records, as a function of the scattering angle relative to the beam direction, the intensity of light scat· A variety of biologically important processes can be tered by the cells. This differential light scattering pattern accurately studied by differential light scattering. Physical embodies a wealth of information about the cell ensemble, ) changes in mitochondria subjected to various enzymes, pH such as cell size. shape, structure, size distribution 10,11, variations, and osmotic stresses can be directly moni· and even structural details such as cell wall thickness and tored1·3.-Systematic distortions of bacterial cells by pre- the refractive indices of the cell wall and cytoplasm 12. A final example of considerable interest concerns the radius of 432 ± 10nm which decreased to 403 ± 10n, .. __, __ ____ effects of heat killing on cell size and size distribution. In 30 minutes heating. The average cell wall thickness rem"~-- preparing autologous staphylococcus vaccines, many labora nearly constant at 108 ± 20nm This is a scanned page of a printed document, likely an order form or information request. The page contains only text, formatted into paragraphs and a form with blank fields for name, title, department, organization, street, city, state, and zip code. There are also lines to indicate the user's interest in light scattering and specific supplements. No photographs, stamps, handwritten notations, diagrams, or redactions are visible. interest concerns the radius of 432 ± 10nm which decreased to 403 ± 10n, .. __, __ ____ effects of heat killing on cell size and size distribution. In 30 minutes heating. The average cell wall thickness rem"~-- preparing autologous staphylococcus vaccines, many labora nearly constant at 108 ± 20nm despite the heating, but th'- breadth of the size distribution increased by 15% after· tories use heat as a sterilization procedure. Such a treatment heating. supposedly does not destroy the immunogenic properties of vaccines and would be expected, therefore, to have little or no effect on cell walls. Figure 7 shows the changes in the References differential light scattering patterns as a function of heating times for S. epidermidis broth suspensions at 60°C. (The curves have been broken at 65° and displaced relative to 1. G. S. Gotterer, T. E. Thompson, and A. L. Lehninger, "Angular light-scattering studies on isolated mitochondria," each other for visual clarity.) A subsequent analysisS of this J. Biophysical and Biochemical Cytology 10, 15 (19611. data showed that the un-heat treated cells had an average 2. L. Packer, "Metabolic and structural states of mitochondria," J. Bioi. Chern. 235, 242 (1960). 3. L. Packer and R. H. Golder, "Correlation of structural and metabolic changes accompanying the addition of carbohy· drates to Ehrlich ascites tumor cells," J. Bioi. Chem. 235, 1234 (1960). 4. R. M. Berkman, ''The effects of formaldehyde, phenol, and other alcohols on bacterial structure deduced from light scattering," Am. Soc. for Microbial. Proceedings, May 1971. 5. R. M. Berkman and P. J. Wyatt, "Differential light scattering measurements of heat treated bacteria," Appl. Microbiology 20, 510 (1970). 6. L. Packer, P. A. Siegenthaler, and P. S. Nobel, "Light induced high amplitude swelling of spinach chloroplasts," Biochem., Biophys. Research Communications 18, 474 (19651. 30omo·icn . . 7. L. Packer, "Structural changes correlated with photochemical phosphorylation in chloroplast membranes," Biochimica et Biophysica Acta 75, 12 (1963). 8. L. Packer, R. H. Marchant, and Y. Mukohata, "Structural changes related to photosynthetic activity in cells and chloroplasts," Biochimica et Biophvsica Acta 75, 23 ( 1963). 10 m1n .. 60'C 9. R. M. Berkman, P. J. Wyatt, and D. T. Phillips, "Rapid detection of penicillin sensitivity in Staphylococcus au reus/' Nature 228, 458 (1970). 10. A. L. Koch, "Theory of the angular dependence of light scattered by bacteria and similar siled biological objects," J. Theoret. Bioi. 18,133 (1968). ' 3 min The document page features a title "Physiological Monitoring of Bacteria and Mitochondria" with accompanying text, including an "Introduction" section. Visually, there are two distinct diagrams: Figure 1, an illustration of a piece of laboratory equipment likely related to optical measurement, and Figure 2, a schematic diagram illustrating a light scattering experiment with labels for "LASER," "CUVETTE," and "DETECTOR," indicating the experimental setup. The top of the page contains a header with the "SCIENCE APPLICATION NOTE" title and contact information, suggesting it's a technical document or publication. No photographs of people, handwritten annotations, or official stamps are visible. The page displays a scientific paper, featuring a line graph labeled "Figure 7" which illustrates differential light scattering patterns for bacterial suspensions. The graph shows curves representing different heating times and temperatures. Surrounding the graph is text that includes a title, references to published studies, and contact information for Science Spectrum, Inc. There are no photographs, stamps, handwritten annotations, or obscured content visible on this page. R. M. Berkman, P. J. Wyatt, and D. T. Phillips, "Rapid detection of penicillin sensitivity in Staphylococcus au reus/' Nature 228, 458 (1970). 10. A. L. Koch, "Theory of the angular dependence of light scattered by bacteria and similar siled biological objects," J. Theoret. Bioi. 18,133 (1968). ' 3 min .. 11. P. J. Wyatt, "Differential light scattering: a physicat'method .Y 60'C for identifying living bacterial cells," Applied Optics 7, 1879 (19681. 12. P. J. Wyatt, "Cell wall thickness, size distribution, refractive Control index ratio, and dry weight content of living bacteria," Nature 226, 277 (19701. 13. Atlas of Light Scattering Curves, (Science Spectrum, Inc. • Santa Barbara, California, 1971 ). 14. T. P. Wallace and J. P. Kratohvil, "Particle size analysis of polymer latices by light scattering", J. Polymer Sci. C, 25, 89 (1968). 15. T. P. Wallace and J. P. Kratohvil, "Size distribution analysis of polymer latex systems by use of extrema in the angular scattering intensity," J. Polymer Sci. A-2, 8, 1425 (1970). 40 60 80 100 16. P. J. Wyatt, "Light scattering in the microbial world," On the SCATTERING ANGLE Occasion of the Centennial of R<>vleigh Scattering Theory, Am. Chern. Soc., Sept. 1971 (to be published in J. Colloid and Interface Science). 17. R. J. Fie!, "Small angle scattering of bioparticles," Experi Figure 7 mental Cell Research 59,413 (1970}. For further information Call or write the Director of Advanced Technology, Science Spectrum, Inc., 1216 State Street, Santa Barbara, California 93105; telephone (805) 963-8605. M1-17·081 Rapid Assay of Bacteria m Urine Introduction LASER CUVETTE f The detection of threshold concentrations of bacteria 0~ in specimen solutions such as urine presents an important )Jo medical challenge. If it were possible to make a rapid deter ):) mination of whether the bacterial count in urine is greater or less than 104/ml (0.1 critical level) 1, it would expedite enormously what is now a very time-consuming procedure. A testament to the urgency of this need is the recent work DETECTOR at NASA2, whose luciferase - ATP assay to detect life on other planets is being considered for detecting bacteria Figure 2 in urine. A more direct bacterial counting capability, one which is simple, effective and rapid, is available via the Differential light scattering patterns can be analyzed technique of laser light scattering using a commercially theoretically using computer software already developed, available table-top instrument, the DIFFERENTIAL I. or simply compared The page features a technical document with a title "APPLICATION NOTE" and a section titled "Rapid Assay of Bacteria in Urine." There are two black and white diagrams: Figure 1 shows a piece of laboratory equipment labeled "DIFFERENTIAL I Photometer," and Figure 2 is a schematic illustrating a laser, cuvette, and detector setup. The text is in columns, and there are no photographs of people, handwritten annotations, or official stamps. There is also no visible evidence of experimental procedures, equipment, or facilities beyond the diagrams of representative equipment. 2 in urine. A more direct bacterial counting capability, one which is simple, effective and rapid, is available via the Differential light scattering patterns can be analyzed technique of laser light scattering using a commercially theoretically using computer software already developed, available table-top instrument, the DIFFERENTIAL I. or simply compared to previously compiled "known" scat tering curves in a pattern recognition approach, analogous to fingerprint identification. An Atlas of Light Scattering DIFFERENTIAL I Photometer Curves5 is available which permits even those not previously familiar with differential light scattering to quickly and accurately determine many of the important physical para meters of cells in suspension. In addition, measured changes in the light scattering pattern can be employed to monitor the effects of variation of conditions (heat, nutrient changes, drug treatment, etc.) on bacterial suspensions. A number of these applications have already been carried out using the Dl FFERENTIAL I instrument4.6,7,8. Urine Specimen Assays The simple task of determining concentrations of bacteria does not need to utilize these analytical aids how ever. In studies of bacterial suspensions using the Figure 1 DIFFERENTIAL I the detection of bacterial concentrations of 105/ml is routine. Indeed, in applications such as anti biotic susceptibility testing, solutions are prepared at about The DIFFERENTIAL I laser light scattering photom this concentration for optimal results. At these concentra· eter is a highly versatile semi-automatic instrument designed tions and lower, the intensity of the scattered light at any to study liquid suspensions of cells with minimum altera angle relative to the background from the liquid system·is tion of their normal environments. The instrument, shown approximately proportional to the number and density of in Fig. 1, records the intensity variation with angle, 8, of the cells, especially when the cell size distribution is narrow. scattered light which results when a cuvette of the solution Thus, calibration of the light scattering patterns in terms of under study is illuminated by a laser beam. The operation is cell concentration is straightforward. shown schematically in Fig. 2. Figure 3 shows a set of light scattering recordings The variation with angle of scattered light intensity is taken on the DIFFERENTIAL I for pure distilled water detected by a specially-designed scanning system which and with several bacterial concentrations as indicated. The records the output on a strip chart or x-y recorder, or on a detectability of these levels can be clearly seen. digital data card punch is taken on the DIFFERENTIAL I for pure distilled water detected by a specially-designed scanning system which and with several bacterial concentrations as indicated. The records the output on a strip chart or x-y recorder, or on a detectability of these levels can be clearly seen. digital data card punch unit. In specimen solutions such as urine, appreciable back When the size and internal structure of the illumi ground light may be scattered from various materials other nated particles have dimensions approximating the wave than the bacteria, materials such as tissue cells, granules, length of the incident light, as do bacteria, the scattered cell debris, leukocytes, erythrocytes and various crystals. To light pattern is particularly sensitive to these particle param gauge the magnitude of this background scattering, bacteria eters. The features (amplitude and angular positions of in known concentrations were added to unprocessed urine maxima and minima) in the scattering pattern give a precise and the samples examined in the DIFFERENTIAL I. Some measure of the size, shaoe, structure. and size distribution 0f th<! rvnif'«l sr:attPrinn n"tt'!r,-.s ?•P <r"'·"" in J=;.., t! It.-~., '>-·--------- I Figure 3 Figure 4 s.aureus in water Bacteria added to urine "'·..._ SCATTEAINQ AHGLE,....,_ 130 150 Figure 5 Clinical urine samples '-------~ 7 x 10 5 ML • ...,.._ ___. ....... __ 1 X 104 ML. ___. ....._ ___. ...,.....,.,._.-...__PURE H0 2 40 SCATTERING ANGLE, degr- able, as expected; even at a lower concentration of 2.8 x 1o 4tml the pattern is emerging from the background signal. These results for untreated urine are very encouraging. Simple techniql:es such as warming, mild acid treatment, and sedimentation all of which are compatible with rapid processing, and should not affect the bacteria, can remove "'-c most of the background-producing material, thereby reduc ing the background scattering levels so that bacterial con centrations appreciably lower than 10 5fml can be measured. Qualitative Studies Figures 5A through 50 show light scattering patterns, SCATTERING ANGLE, degrees taken with the DIFFERENTIAL I, based on four patient urine samples9. The protocol for all four was: References 1 loopfull of urine was placed in trypti case broth and 1. R. N. Barnett, "Conference on the Medical Usefulness of Micro incubated for six hours; biology," Amer. J. of Clinical Pathology 54 Part II, 521 (1970). 2. G. L. Picciolo, B. N. Kelbaugh, E. W. Chappelle, A. J. Fleig," An • 0.5 ml of the incubated This page contains three scientific graphs illustrating light scattering patterns from bacterial samples and clinical urine samples. Figure 3 shows the light scattering of *S. aureus* in water at different concentrations, while Figure 4 displays bacteria added to urine. Figure 5 presents clinical urine samples, comparing a sample with and without colimycin. The page also includes a list of nine scientific references and contact information for Science Spectrum, Inc. There are no photographs, handwritten annotations, official stamps, forms, or handwritten marginalia visible on this page. The document does not contain any redacted content. trypti case broth and 1. R. N. Barnett, "Conference on the Medical Usefulness of Micro incubated for six hours; biology," Amer. J. of Clinical Pathology 54 Part II, 521 (1970). 2. G. L. Picciolo, B. N. Kelbaugh, E. W. Chappelle, A. J. Fleig," An • 0.5 ml of the incubated solution was placed in 13.5 Automatic Luciferase Assay in Bacteria in Urine," NASA ml of distilled water in a cuvette; Goddard Report X-641-71-163 (Apr. 1971). • the cuvette was placed in the DIFFERENTIAL I and 3. A. L. Koch. "Theory of the Angular Dependence of Lig