Klett-Summerson colorimeter

Fig. S. Converting Klett units to number of cells per milliliter. A blue filter was used in the Klett-Summerson colorimeter, and the cells were E. coli. Zero units were set with Nephlo flasks containing the growth medium (0.2% glucose in Davis salts) without cells. The culture was started with an inoculation that gave 5-8 X 106 cells/ml. The chart is most accurate between 15 and 170 units.

Standard dye solution 10 mg of 2, 6 - dichlorophenol -indophenol (DCPIP) (BDH) was dissolved in deionized water at 80 C, cooled, and its volume made upto 200 ml. Its strength was standardized by the addition of either deionized water or more concentrated dye solution until 5 ml of the dye solution when mixed with 2 ml of deionized water, gave a reading of 390 on the scale of the Klett-Summerson colorimeter or an optical density (O.D.) of 0.78. This standardization of the dye solution was done every time Just prior to starting of estimations. 

One ml aliquot of each AA solution was mixed with 1.0 ml of buffered HPO solution and 5 ml of standardized dye solution and the readings were recorded on a Klett-Summerson colorimeter using a green filter. Ten to 20 replicates were taken for each concentration of AA solution and these were converted into O.D. by multiplying with 0.002 (calibration factor of Klett Summerson colorimeter). 

Calculation The colorimetric reading was converted into optical density (O.D.) by multiplying with 0.002 (the calibration factor of Klett Summerson colorimeter). Using the regression formula, Y 0.1103 - 0.14 X, the concentration of AA in one ml of the original extract and then AA/g fresh weight of the material was calculated. 

KU, Klett units (after turbidity measurement in a Klett-Summerson colorimeter and calculating final reading times the dilution). 

Shown in Fig. 6.8 is an early electronic Klett-Summerson colorimeter that was a step above the color comparator shown above. With this instrument the analyst could now abandon the unreliable human eye as detector and begin to rely on a variety of detection and signal recording methods. We have moved a long way from Duboscq and Klett, and yet these are the instruments that started us down the pathway of rapid, accurate, sensitive color measurement for a variety of purposes. 

Colorimetry Measurements. A Klett-Summerson photoelectric colorimeter. Model M3896, equipped with a No. 42 filter having an approximate spectral range from 400 to 465 m/x, was used for colorimetric measurements. 

KU = Klett Units, the optical density units measured by Klett-Summerson photoelectric colorimeter. 

Development of Turbidity. The tubes are removed from the bath at 0.5 minute intervals and 4 ml. of the acidified serum-buffer mixture is added immediately. The tubes are shaken and allowed to stand at room temperature for 30 minutes. Turbidities are then read in a suitable photoelectric colorimeter. (For the Klett-Summerson instrument, the blue filter is recommended by the manufacturer.) The zero reading of the instrument is set with the blank tube containing 1 ml. of pH 6 buffer and 4 ml. of acidified serum-buffer mixture. The absorption of the Klett tubes varies as much as 10 to 15 Klett units. The tubes should be calibrated with... 

Fig. 6.8 Klett-Summerson photoelectric colorimeter, clinical model. Photo by Gregory Tobias. Courtesy of the Chemical Heritage Foundation Collections...

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