4.2. DETERMINACION DEL NIVEL DE CONOCIMIENTO EN MATERIA TRIBUTARIA
4.2.4. GRADO DE CONOCIMIENTO Y CONDUCTAS REFERIDAS A LOS
I
1
o 0.50 0.45 0.40 0.35 0.30 % 0.25 S ÜÛ 0.20 B ^ 0.15 0.10 0.05 2 4 6 8 10 12 14 Particle midsize (^m)Approximately 50 mL of cells in liquid suspension were released into the top of the Bioaerosol Test Chamber using the atomiser. The Aerodynamic Particle Sizer sampled air from the bottom of the chamber.
Luria broth only, 3.8 x 10* cells.mL"\ 3.8 x 10^ cells.mL"\ 3.8 x 10’° cells.mL"'
Chapter Three: Mass Balancing in Contained Environments 3.6.2.1 Aerosol Masses
Bennett and Norris (1989) showed that the deposition and size distribution of aerosol particles depend upon the concentration of dissolved solids in the original suspension. The possiblity of a microbial cell occurring in each airborne particle will depend upon the concentration of microorganisms in the aqueous suspension.
Hambleton et al. (1992) discuss the particle size distribution of aerosols produced from liquid suspensions. The ultimate particle size of the droplet will depend not only on the initial droplet size, but also on the mass concentration of the original suspension. This includes the solution that the cells are dispersed in, as well as the cells themselves.
Tables 3.25 and 3.26, below, show aerosol masses calculated for each concentration for both cell species. This mass includes both the mass of the cells and of the suspending solution.
Cell volume = 7id^
6
Where d = 5|xm = 5 x 10"^ m for S. cerevisiae (1 x 10"^ m for E. coli)
Volume - 71X T5 X = 6.545 x lO'^^ m^
6
Density = 1400 kg.m"^
Therefore mass of one yeast cell = 6.545 x 10"^^ x 1400 = 9.163 x 10" kg
If there are 2.4 x 10^ cells.mL" mass of ImL = 9.163 x 10"^^ x 2.4 x 10^ = 2.2 mg.mL"!
Malt extract or Luria broth is added at a concentration of 20g.L"l, therefore 20mg.mL"l.
Tables 3.25 and 3.26 below show that the broth present in the suspensions makes up the majority of the total mass at the lower cell concentrations. It is only when there is more than 10^ cells.mL" 1 present that the cell mass becomes the larger factor.
Chapter Three: Mass Balancing in Contained Environments Table 3.25: Mass Concentration of S. cerevisiae Suspensions
S. cerevisiae concentration (cells.mL"^)
Mass of cells only (mg.mL-1)
Mass of cells and broth (mg.mL'l)
2 . 4 x 10'7 2.3 22
2.4 X 10^ 23 43
2.1 X 109 192 212
Table 3.26: Mass Concentration of E. coli Suspensions
E. coli concentration (cells.mL"^)
Mass of cells only (mg.mL-l)
Mass of cells and broth (mg.niL"l)
3.8 X 1q8 0.28 20.3
3 .8 x 1q9 2.8 22.8
3 . 8 x IQIO 28 47.8
3.Ô.2.2 Particle Size Distribution and Cell Recovery in the Cyclone
This particle size distribution study was carried out to discover if there was a difference in the particle size of aerosols produced from cell suspensions of different concentration. It was thought that this may be the reason for a decrease in cell recovery in the cyclone as released cell concentration increased. Larger diameter particles would fall to the floor of the cabinet more easily and evade capture in the cyclone. If more of these larger particles were produced from the higher cell concentrations this would explain the reduced cyclone recovery.
Figures 3.4 and 3.5 show more smaller particles (3-8 pm) present at the higher concentration of both S. cerevisiae samples at the highest cell concentration only. At the larger particle diameters there is a less marked difference in the mass of particles present at the higher S. cerevisiae concentrations. This does not correlate with the cyclone recoveries for S. cerevisiae cells in both broth and Ringers'
Chapter Three: Mass Balancing in Contained Environments
solution (tables 3.15 and 3.16), which decrease as released cell concentration increases.
The particle size distributions of the highest cell concentration for E. coli aerosols (figures 3.6 and 3.7) show a larger number of all particles sizes. This is more marked for E. coli suspended in Ringers' solution rather than fermenter broth. Again, these particle size distributions do not correlate well with the cyclone recovery data in tables 3.17 and 3.18. Table 3.17 shows that recovery of E. coli
suspended in Ringers' solution in the cyclone increase as released cell concentration increases. Table 3.18, shows that recovery in the cyclone of E. coli
suspended in fermenter broth decreases as released cell concentration increases. This data are discussed further in section 5.2.
3.7 The Polymerase Chain Reaction
3.7.1 Detection of Transketolase E, coli Cells Using Kanamycin Primers
These experiments were carried out to investigate:
1. Whether Transketolase (TK) E. coli cells could be detected in the presence of unmodified (UM) E. coli cells.
2. The limit of detection of the Polymerase Chain Reaction (PGR) to TK E. coli
cells.
Both TK and UM E. coli cells were grown up in shake flasks containing Luria broth and Nutrient broth (with Kanamycin) respectively. At harvest, the TK E. coli
had reached a concentration of 2.9 x 10^ CFU.mL'^ and the UM had reached a concentration of 3.6 x 10^ cells.mL"^. The two fermentation broths were then diluted from 10^ CFU.mL"! to 10^ CFU.mL'l in sterile RO water. Each of the 6
TK E. coli dilutions was prepared using one of the following UM E. coli
concentrations as a diluant: 10^, 10^, 10^ and 10^ CFU.mL'^.
PGR was carried out on these 24 combinations using the KM l and KM2 primers (see section 2.3.4.3 for method). The PGR products were loaded on electrophoretic gels shown in figures 3.8(i) and (ii).
Chapter Three: Mass Balancing in Contained Environments
This experiment demonstrates that, using PCR, it is possible to detect 2.9 x 10^ TK
E. coll cells, in a background of 3.2 x 10^ UM E. coli cells. This is equivalent to 1 TK E. coli cell in 10^ UM E. coli cells.
The pUC plasmid, which contains the DNA fragment recognised by KM l and 2, has a copy number o f 2-300 per cell. It is therefore not unrealistic to expect to detect less than 10^ target cells. One way of improving the sensitivity of the PCR reaction may be to lyse the target cells before carrying out the reaction.
Figure 3.9 shows the electrophoretic gel for the PCR products produced when the cell suspensions are heated to 99®C for 30 minutes before carrying out the PCR. It can be seen that 2.9 x 10^ TK E. coli cells can be detected in a background of 3.2 x 10^ UM E. coli cells. Thus, a lysis step before carrying out the PCR improved the sensitivity 10-fold. However, there was no improvement in the sensitivity of the PCR for TK E. coli cells in the absence of UM E. coli cells.
Chapter Three: Mass Balancing in Contained Environments