Introduction

All of the above methods either require the availability of expensive equipment or the substantial investment of time. In reality, the most often used method simply monitors the optical density of the sample. The absorbance of the sample measured in a spectrophotometer is correlated to either the dry weight or the number of cells per volume.

Biomass concentration is one of the most critically needed measurements in fermentation studies. It is also one of the most difficult and unreliable ones. For example, all the above dry/wet weight methods and all the automated counting equipment fail completely if the broth contains other insoluble particulate matter, which is often the case in a practical fermentor. Similarly, the optical density measurement only has limited usefulness if the fermentation broth is not clear. In addition, these methods cannot distinguish the viable cells from the dead ones. On the other hand, the standard plate count can detect viable cells among other particulate matters. However, the method requires elaborate preparations, and it takes 24-48 hours for the cells to be incubated and counted; the cost of Petri dishes and media can also be prohibitive. Consequently, the direct plate count is useless in feedback control of a fermentation process; it is mainly used industrially to countercheck other measurements, especially the optical density.

In this experiment, the cell density of a given sample will be measured with the following five methods: wet weight, dry weight, optical density, direct cell counting with a chamber, and successive dilutions followed by plating.

List of Reagents and Instruments

A. Equipment

• Flasks
• Graduated cylinder
• Filtration unit with vacuum pump
• Filter membrane or weighing pan
• Centrifuge
• Oven, 100 ºC
• Balance
• Spectrophotometer
• Cell counting chamber
• Microscope
• Petri dish, 4 per standard plate count
• Sterile pipets, 4 per standard plate count
• Sterile bottles, 3 per standard plate count

B. Reagents

• Flask of culture
• Nutrient (YPG) agar
• Sterile water

Procedures

1. Dry/Wet Weight Measurement:
   • Dry in an oven an empty aluminum weighing pan or a sheet of cellulose acetate filter membrane, 47mm in diameter, 0.45µm in pore size. Weigh them and store them in a desiccator lined with Drierite (anhydrous CaSO₄).
   • Stir the flask to suspend the culture evenly. Pour out 100 ml of the culture into a graduated cylinder.
   • Separate the cells from the broth either by centrifugation at 10,000 g for 5 minutes or by filtration. In the case of centrifugation, carefully discard the clear broth and scrape the cell paste from the centrifuge tube into a weighing pan. Rinse the centrifuge tube with a few ml of water. Pour the rinse water into the weighing pan, as well. In the case of filtration, the culture is poured into the holding reservoir fitted on the filter membrane. A vacuum is applied to pull the liquid through the membrane. Rinse the reservoir with a few ml of water and scrape any paste adhering to the glassware. The wet weight of the culture is measured immediately after all the water has been pulled through.
   • Dry the cell paste in an oven set at 100ºC. The cells will be charred and the filter membrane will be burned if the temperature of the oven is set too high. Measure the weight of the pan/filter plus the cell paste periodically until there is no further decrease in the dry weight. It will take 6-24 hours to dry the sample completely, depending on the oven temperature and the thickness of the paste. Calculate the difference in the weight, and express the dry weight in g/l.
2. Optical Density:
   Dilute the sample to appropriate concentrations as needed, and measure the absorbance of the sample with a spectrophotometer at 550 nm. Other wavelengths may also be used, but one must be consistent. Generate a calibration curve to relate the absorbance with cell dry weight. The usual rules of operating a spectrophotometer apply here, as well. For example, the accuracy of the method is the highest when the absorbance is between 0.1 and 0.5. For a given culture sample, a good spectrophotometer should yield a linear relationship between the number of cells and the absorbance. However, the optical density is also a function of cell morphology such as size and shape, because the amount of transmitted or scattered light depends strongly on these factors. Consequently, an independent calibration curve is required for each condition in accurate research work, as the cell size and shape depend on the specific growth rate and the nutrient composition. As a rule of thumb, an optical density of 1 unit corresponds to approximately 1 g/l of dry cell. This is also commonly referred to as the turbidity measurement.
3. Cell Counting Chamber:
   This is a standard method for counting the number of microorganisms in milk. It is also widely used in blood counts and vaccine counts; thus, a counting chamber is also commonly called a hemocytometer. But the technique is not very popular among biochemical engineers. Two thin rails of a well defined height of 0.02mm are attached to the surface of a glass slide that is marked with evenly spaced lines at 0.05mm intervals. Thus, each square represents a volume of 5.0X10^{^-8}ml.
   • Add 0.1 ml of methylene blue and 1 ml of the suspended culture to a test tube. Mix well. Cells are stained for 3 minutes to enhance visualization. Add water to dilute the sample as needed. This introduces a dilution factor which must later be incorporated into the calculation of the original cell density.
   • Clean all the grease from the counting chamber with ethanol so that cells can be clearly counted later.
   • Place a piece of reinforced cover glass on top of the rail. There should be a small space, 0.02 mm to be exact, between the platform and the cover glass.
   • Fill a capillary pipet with the stained sample. Gently touching the tip of the pipet against the edge of the cover glass will attract the sample to fill the space under the cover glass solely by capillary actions.
   • The number of cells enclosed in each square is counted visually under an ordinary light transmission microscope. One may need to raise the oil immersion lens slightly to shift focus at the space under the cover glass. Repeat for at least 20 squares. Take the average number of cells per square.
   • Calculate the number of cells per ml by multiplying the number of cells per square by the dilution factor introduced in Step 3a by 1/(5X10^{^-8}), or 2X10^{^7}.
4. Standard Plate Count:
   The major part of the procedure deals with a series of successive dilutions of the original culture in sterile bottles with sterile water. The diluted culture is poured into Petri dishes along with the nutrient agar. The number of colonies is counted after incubation.
   • Shake the flask containing the culture, and pipet 1 ml of the culture aseptically into a capped sterile bottle marked "A," which contains 99 ml of sterile water. Vigorously shake the bottle A to mix the culture and break any flocculating clumps of microorganisms. The dilution factor in bottle A is 1:100. A dilution factor of 1:101 is often more easy to work with physically than 1:100; in this case, the subsequent dilution factor is also similarly modified without any loss in the accuracy.
   • With the second sterile pipet, transfer 1 ml from the bottle A into a similar bottle marked "B," which also contains 99 ml of sterile water. Shake and mix. The dilution factor in bottle B is 1:10,000.
   • With the third sterile pipet, transfer 1 ml from the bottle B to a Petri dish marked 1:10,000. Thus, the number of colonies from this dish is multiplied by 10^{^4} to give the number of cells in 1 ml of the original sample.
   • With the same pipet as Step 4c, transfer 0.1 ml from the bottle B to a Petri dish marked 1:100,000. The number of colonies from this dish is multiplied by 10^{^5} to give the number of cells in 1 ml of the original sample.
   • With the same pipet as Step 4c, transfer 1 ml from the bottle B into a third bottle marked "C," which contains 99 ml of sterile water. Shake and mix. The dilution factor in bottle C is 1:1,000,000.
   • With the fourth sterile pipet, transfer 1 ml from the bottle C to a Petri dish marked 1:1,000,000. Thus, the number of colonies from this dish is multiplied by 10^{^6} to give the number of cells in 1 ml of the original sample.
   • With the same pipet as Step 4f, transfer 0.1 ml from the bottle B to a Petri dish marked 1:10,000,000. The number of colonies from this dish is multiplied by 10^{^7} to give the number of cells in 1 ml of the original sample.
   • Heat the capped culture tube containing 50 ml of agar to boil for 10 minutes. The heating both melts and sterilizes the agar.
   • After the agar is cooled to 45ºC, pour 12 ml to each of the four Petri dishes. The culture will be killed if the agar is too hot; it will solidify if it is cooled for too long. Swirl the plates very gently to mix the culture with the agar.
   • Allow the agar to solidify.
   • Incubate the plates in the inverted position at 37ºC for 48 hours.
   • Select those plates that have 30-300 colonies. Count every colony, large and small. To keep track of the counted colonies, dot the colonies with a permanent marker pen as one counts them. Different colored pens may be used for a mixed culture.

     Statistically, the most reliable results are given by plates with between 30 and 300 colonies. Only about two significant figures can be obtained from this method. The accuracy can be improved if multiple plates can be prepared. This, however, is rarely done due to the cost.

   • Some of the plating may be omitted if the number of cells per ml can be estimated to the order of magnitude. Finally, a cell counting chamber can be used in conjunction with cell plating to distinguish the number of viable and nonviable cells.

Questions

1. Report the cell density in appropriate units for the given sample.