Materials:
4 culture plates Quebec colony counter mechanical hand counter felt pen (optional)
Bacterial Population Counts • Exercise 23
Figure 23.3 Standard procedure for shaking water blanks requires elbow to remain fixed on table
Figure 23.4 Colony counts are made on a Quebec counter, using a mechanical hand tally
Benson: Microbiological Applications Lab Manual, Eighth Edition
IV. Culture Methods 23. Bacterial Population Counts
© The McGraw−Hill Companies, 2001
1. Lay out the plates on the table in order of dilution and compare them. Select the plates that have no fewer than 30 nor more than 300 colonies for your count. Plates with less than 30 or more than 300 colonies are statistically unreliable.
2. Place the plate on the Quebec colony counter with the lid removed. See figure 23.4. Start counting at the top of the plate, using the grid lines to prevent counting the same colony twice.
Use a mechanical hand counter. Count every colony, regardless of how small or insignificant.
Record counts on the table in section A of the Laboratory Report.
Alternative Counting Method: Another way to do the count is to remove the lid and place the plate upside down on the colony counter.
Instead of using the grid to keep track, use a felt pen to mark off each colony as you do the count.
3. Calculate the number of bacteria per ml of undi-luted culture using the data recorded in section A of the Laboratory Report. Multiply the number of colonies counted by the dilution factor (the recip-rocal of the dilution).
Example: If you counted 220 colonies on the plate that received 1.0 ml of the 1:1,000,000 di-lution: 220 ⫻ 1,000,000 (or 2.2 ⫻ 108) bacteria per ml. If 220 colonies were counted on the plate that received 0.1 ml of the 1:1,000,000 dilution, then the above results would be multiplied by 10 to convert from number of bacteria per 0.1 ml to number of bacteria per 1.0 ml (2,200,000,000, or 2.2 ⫻ 109).
Use only two significant figures. If the num-ber of bacteria per ml was calculated to be 227,000,000, it should be recorded as 230,000,000, or 2.3 ⫻ 108.
T
URBIDIMETRYD
ETERMINATIONS When it is necessary to make bacteriological counts on large numbers of cultures, the quantitative plate count method becomes a rather cumbersome tool. It not only takes a considerable amount of glassware and media, but it is also time-consuming. A much faster method is to measure the turbidity of the culture with a spectrophotometer and translate this into the number of organisms. To accomplish this, however, the plate count must be used to establish the count for one culture of known turbidity.To understand how a spectrophotometer works, it is necessary, first, to recognize the fact that a culture of bacteria acts as a colloidal suspension, which will intercept the light as it passes through. Within certain limits the amount of light that is absorbed is directly proportional to the concentration of cells.
Figure 23.5 illustrates the path of light through a spectrophotometer. Note that a beam of white light passes through two lenses and an entrance slit into a diffraction grating that disperses the light into hori-zontal beams of all colors of the spectrum. Short wavelengths (violet and ultraviolet) are at one end and long wavelengths (red and infrared) are at the other end. The spectrum of light falls on a dark screen with a slit (exit slit) cut in it. Only that por-Exercise 23 • Bacterial Population Counts
Figure 23.5 Schematic of a spectrophotometer
tion of the spectrum that happens to fall on the slit goes through into the sample. It will be a monochro-matic beam of light. By turning a wavelength control knob on the instrument, the diffraction grating can be reoriented to allow different wavelengths to pass through the slit. The light that passes through the culture activates a phototube, which, in turn, regis-ters percent transmittance (% T) on a galvanome-ter. The higher the percent transmittance, the fewer are the cells in suspension.
There should be a direct proportional relationship between the concentration of bacterial cells and the absorbance (optical density, O.D.) of the culture. To demonstrate this principle, you will measure the %T of various dilutions of the culture provided to you.
These values will be converted to O.D. and plotted on a graph as a function of culture dilution. You may find that there is a linear relationship between concentra-tion of cells and O.D. only up to a certain O.D. At higher O.D. values the relationship may not be linear.
That is, for a doubling in cell concentration, there may be less than a doubling in O.D.
Materials:
broth culture of E. coli (same one as used for plate count)
spectrophotometer cuvettes (2 per student)
4 small test tubes and test-tube rack 5 ml pipettes
bottle of sterile nutrient broth (20 ml per student)
1. Calibrate the spectrophotometer, using the proce-dure described in figure 23.7. These instructions are specifically for the Bausch and Lomb Spectronic 20. In handling the cuvettes, keep the following points in mind:
a. Rinse the cuvette several times with distilled water to get it clean before using.
b. Keep the lower part of the cuvette spotlessly clean by keeping it free of liquids, smudges, and fingerprints. Wipe it clean with Kimwipes or some other lint-free tissue. Don’t wipe the cuvettes with towels or handkerchiefs.
c. Insert the cuvette into the sample holder with its index line registered with the index line on the holder.
d. After the cuvette is seated, line up the index lines exactly.
e. Handle these tubes with great care. They are expensive.
2. Label a cuvette 1:1 (near top of tube) and four test tubes 1:2, 1:4, 1:8, and 1:16. These tubes will be used for the serial dilutions shown in figure 23.6.
3. With a 5 ml pipette, dispense 4 ml of sterile nutri-ent broth into tubes 1:2, 1:4, 1:8, and 1:16.
4. Shake the culture of E. coli vigorously to suspend the organisms, and with the same 5 ml pipette, transfer 4 ml to the 1:1 cuvette and 4 ml to the 1:2 test tube.
5. Mix the contents in the 1:2 tube by drawing the mixture up into the pipette and discharging it into the tube three times.
6. Transfer 4 ml from the 1:2 tube to the 1:4 tube, mix three times, and go on to the other tubes in a similar manner. Tube 1:16 will have 8 ml of di-luted organisms.
7. Measure the percent transmittance of each of the five tubes, starting with the 1:16 tube first. The contents of each of the test tubes must be trans-ferred to a cuvette for measurement. Be sure to close the lid on the sample holder when making measurements. A single cuvette can be used for all the measurements.
8. Convert the percent transmittance values to opti-cal density (O.D.) using the following formula:
O.D. ⫽ 2 ⫺ log of percent transmittance Example: If the percent transmittance of one of your dilutions is 53.5, you would solve the prob-lem in this way:
Bacterial Population Counts • Exercise 23
Figure 23.6 Dilution procedure for cuvettes
Benson: Microbiological Applications Lab Manual, Eighth Edition
IV. Culture Methods 23. Bacterial Population Counts
© The McGraw−Hill Companies, 2001
O.D. ⫽ 2 ⫺ log of 53.5
⫽ 2 ⫺ 1.7284
⫽ 0.272
Table II of Appendix A is a log table. Of course, if you have a calculator, all this is much simpler.
Logarithm Refresher In case you have forgotten how to use logarithms, recall these facts:
Mantissa: The value you find in the log table (0.7284 in the above example) is the mantissa.
Characteristic: The number to the left of the dec-imal (1 in the example) is the characteristic.
This figure (the characteristic) is always one number less than the number of digits of the figure you are looking up.
Examples:
number characteristic mantissa
5.31 0 .7251
531 2 .7251
Although the galvanometer may show absorbance (O.D.) values, greater accuracy will result from calculating them from percent transmittance.
9. Record the O.D. values in the table of the Laboratory Report.
10. Plot the O.D. values on the graph of the Laboratory Report.
Exercise 23 • Bacterial Population Counts
1
Turn on instrument by rotating zero control knob clockwise. Do this 20 minutes before measurements are to be made. Also, set wave-length knob (top of instrument) at 686 nanometers wavelength. Adjust the meter needle to zero by rotating zero control knob.2
Insert a cuvette containing 3 ml of sterile nutrient broth into sample holder. The cover must be closed.Keep the index line of cuvette in line with index line on the sample holder.
Refer to instructions 1a through 1e on page 97 concerning care of cuvette.
3
Adjust the meter to read 100%transmittance by rotating light–
control knob. Remove cuvette of nutrient broth and close lid. If needle does not return to zero, readjust accordingly. Reinsert nutrient broth again to see if 100% transmittance still registers. If it has changed, re-adjust with light–control knob. Once meter is adjusted for 0 and 100%, transmittance, turbidity measurements can be made. Recheck calibration from time to time to make certain instrument is set properly.
Figure 23.7 Calibration procedure for the B & L Spectronic 2 on page 97