APPRECIATION OF AN COMPLEX
ULTRASOUND SYSTEM ACCORDING TO SURVIVAL CELL COUNT
M. NEMÉNYI – A. LŐRINCZ
University of West
Hungary, Faculty of Agricultural and Food sciences, Mosonmagyaróvár;
Institute of
Agricultural, Food and Environmental Engineering
Summary
The decreasing of
micro-organism cell count the most important task of the food industry. The
killing of microbe no too difficult thing rather the preserve of essential food
components is the heavey work. The modern microbe destructor technologyes its
effects goals only the pointed objects and dont touchs the other essential food
components. Our experimental work goals this philosophy with ultrasound. We
examinated the surviving cell count of Saccharomyces
cerevisiae suspension. This cell suspension was of an closed liquid circuit
system streaming with peristaltic pump. The cell count with manual and
automatical detection system was examinated and the experimental method based
on vital staining with methylen blue and on the cell counting. The results
gives possibility any cell phase analytical and continuous cell decreation
system developing. This work is fundamental research.
There is no cavitation in the ultrasound field
until the amplitude of the acoustic pressure exceeds a certain level, the cavitation
threshold [1]. Cavitation threshold is proportional with the frequency of
ultrasound, with the hydrostatic pressure in the liquid, and with the viscosity
of the sample and it is inversely proportional with the gas content and temperature of the sample
[2]. There are two types of cavitation that are stable and transient
cavitation [3]. Basically two reactions take place
when ultrasound and a media interact with each other. One of them is the
absorption the other one is the scattering, which changes e.g., the speed of
propagation of the sound in the subject media [4]. Due to the absorption, the
intensity of ultrasound decreases exponentially with distance and the
absorption coefficient primarily depends on the speed of propagation of the
sound in the subject media, on the wave type, on the material situated in the
ultrasound field and on the frequency. The absorption always characterizes a
media, a structure or an environment that determines the parameters of
propagation [5]. When absorption coefficients were measured in oxo- and és
methemoglobin, it was observed that the absorption is proportional with the
concentration of hemoglobin in the concentration range between 0 and 15
[g/100ml] [6]. It was clearly
established that the profile of the ultrasound propagation speed depends
on the concentration profile of the suspension [7]. Effects of the size and
concentration of the suspended particles on the propagation speed of ultrasound
was examined in water based suspensions. It was established that the speed of
sound largely depended on the particle size and concentration [8]. In vitro
cavitation threshold measurements were carried out in human blood. In the fresh
blood that contained every blood component, the amplitude of the acoustic
pressure belonging to the cavitation threshold was higher than in diluted blood
[9]. Due to cavitation caused by ultrasound, acoustic streaming was formed in
the liquid [10]. Acoustic streaming is a movement of the liquid that is caused
by intensive ultrasound [11]. Mixing of liquid was experienced in the
ultrasound field due to acoustic streaming [12]. An acoustic reflector placed
opposite to the transducer causes a standing wave to be formed. In a standing
wave the materials whose density are lower and higher than of the liquid drift
to propagation cluster planes (pressure antinodes), and pressure nodes,
respectively [2]. The ultrasonic separation is used in analytical biotechnology
applications. This procedure is based on the fact that in a standing wave
field, where there is no cavitation,
the cells are arranged in bands distances of which are smaller than a
millimeter and they can be separated from these bands [13]. Yeast (Saccharomyces
cerevisiae) and rubber particles were manipulated in a standing wave
ultrasound field at frequencies of 1 and 3 [MHz]. The particles formed bands in
pressure nodes whose distance from each other was equal to half of the
wavelength. In the direction of the radiation the bands formed column like
structures. Stability of the bands, the conditions under which they are broken
and the formation of the acoustic streaming were investigated in [14].
Effectiveness of the cell separation of Escherichia coli bacteria and Saccharomyces
cerevisiae yeast cells from a yeast suspension was examined at frequencies
of 1 and 3 [MHz] [16].
Materials and methods
As experimental
marker microorganism we suspended 1,8 gram yeast (Saccaromyces cerevisiae) in 200 cm³ distilled water with a magnetic
mixer until it became clod free and the cell concentration reached the level of
9x107/ml. For the sake of a better detection we put 5 drops of
methylene blue into the solution, which did not influence the vitality of the
microorganism.
We put the suspension into a fluid flow system (Fig. 1.) of 116 cm3
of inner volume with a peristaltic pump. After the filling and short-circuit of
the system the suspension was circulated by a peristaltic pump between the
different structural units. The suspension was then not directly treated but
isolated from its environment by material flow through ultrasonic flow cuvettes
(Fig. 2.) especially made for this purpose. The ultrasonic cuvettes allow the
suspension to flow with a surface of 1 cm2 and a thickness of 0.5
mm. There were two cuvettes placed 1 cm apart at right angles to the flow
direction. The reason for this arrangement was that the effects on the liquid
film are much easier to observe than inside the material. In order to avoid cell sedimentation an efficiently high rate
of flow was applied: 50-70 cm/sec (4-5 cm3/sec). The suspension
flowing in the ultrasonic cuvettes were exposed at a frequency of 0.8 MHz and
at a capacity of 10 W/cm2.
The suspension flowing in the system gets into an optical detection cell
placed in a biological microscope. The picture gets then from here through a
CCD camera into a computer system, where it will be saved according to time
units. Which will allow evaluating the cell disruption effect of the treatment
based on calibration. After short-circuit the flow system and turning on the
ultrasonic system there were drop samples taken at time units through a
built-in tap. The samples were immediately analyzed under a Bürker
chamber. A thermostat unit also belongs
to the system, which ensures a constant temperature for the reproduction of the
tests.
The survive cell analysis is based on a vital stain, which means that
under microscope with Bürker-chamber. The dead cells are stained blue owing to
methylene blue but the living organisms remain clear. We can establish the
curve of deteriorated and survived cells owing to ultrasonic treatment as blue
stained and clear cells are counted at regular time units. Organisms are
regarded as cells, which have intact
cell walls and reflect vitality. We want to mention this fact because after a
certain time of treatment cell lysis will happen.
Treatment
definition
The treatment
means a certain period of time during which the amount of liquid circulating in
a flow system is exposed to a physical (ultrasonic) treatment of a given
capacity during a given period.
The time of
treatment means in the flow system the period between the turning on and
turning off the ultrasound. This period was taken into account during the
evaluation of the results.
As for the
amount of liquid the time of treatment had to be corrected in the flow system
concerning the total amount of liquid flowing in the system and the total time
of treatment. Therefore the total liquid treatment time of „A” is required to reduce the original cell count to its
hundredth where „B”=116cm3 is the total amount of flowing liquid in our system. So 1 cm3
of cell suspension has to be treated at „A/B” minute.
Results and discussion
Treatments were
carried out at 0,8 MHz and with a capacity of 7,5 W/cm2; 9,6 W/cm2;
10,5 W/cm2 and 12 W/cm2 by taking samples from the
suspension at defined times. These samples were then evaluated in Bürker
chambers based on the average living and dead cell numbers to be observed.
Figure 3 showed the relative percentage of survive cell counts in the samples.
According our examinations the relative surviving cell counts of one milliliter
treated suspension showed on table 1. On fig. 3. the ♦ = 7,5 W/cm2,
■ = 9,6 W/cm2, ∆ = 10,5 W/cm2, X = 12 W/cm2
are the points belonging to the ultrasonic treatments and the functions fitted to them (Fig 4.). The relative living
cell counts signified by the different symbols show that more drastic and
faster cell destruction is to be observed depending on the time passed if we
applied higher capacities. In table 1 the relative cell counts refer to 22
cells per Bürker chamber owing to creating the model function, which indicate
an initial cell count of 88 million/millilitre. In order to set up a model formula we set different trend
functions onto the points. The logarithmic trend functions showed the highest
correlation with the measured points. Setting trend functions on to the
additive and multiplying factors of the logarithmic functions resulted in the
formula shown in table 2. where „a” is the trend function referring to the
additive factor and „b” is the trend function referring to the multiplying
factor. Cell count can be omitted from
the model formula as it refers to the initial cell count of 22 cells/Bürker
chamber (cfu), so it has to be corrected by the actual cell counts
respectively.
This model function helps to calculate the ultrasonic capacity that is required
for treating the material at a given initial cell count, so that it would
reduce to the required cell count after a certain period of time. Or it allows
determining the time needed to reach the required cell count at constant
ultrasonic capacity. Of course these values refer to a given system. Therefore
a correction factor is to be used for a general usage adapted to the actual
conditions. Figure 5. shows the
replacement of the treatment of 7.5 W/cm2.
Conclusion
The model
function allows modelling the effects of the ultrasonic treatment at a
frequency of around 0,8 MHz and different capacities.
The ultrasonic treatment
can be adapted for use for treating agricultural products if high quality is
required.
Acknowledgement
We express out thanks for the help of Prof. Dr. Pál Greguss from the
Technical and Economic University of Budapest.
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Figure 1. The complete flow injection system. Key: 1. Peristartic pump, 2. Lead
out tube, 3. Ultrasonic treating cuvettes, 4. Tube, 5. Tap 6. Optical flow
cuvette, 7. Lead in tube, 8. Stereomicroscope, 9. CCD camera, 10. Ultrasound transmitter,
11. Ultrasound receiver, 12. Echoless watertank.
Figure 2. The ultrasound system. Key:
1. Signal generator, 2. Electronic cable, 3. Ultrasound amplifier, 4. Electronic
cable, 5. Ultrasound transmitter, 6. Reflection surface, 7. Ultrasound
receiver, 9. Electronic cable, 9. Oscilloscope, 10. Echoless watertank.
Table 1. Relative surviver cell cell
counts of one millilitre treated cell suspension after different exposition
times.
Ultrasound power Exposition time |
7,5
[W/cm2] 4 min
3 sec |
9,6
[W/cm2] 2 min
51 sec |
10,5
[W/cm2] 2 min
39 sec |
12
[W/cm2] 2 min
30 sec |
Rel surv. Cell count |
1,7% |
0,9% |
0% |
0,8% |
Figure 3. Relative survival
cell count (%)
y=a*lnx0+b
(x0=time,
[min]) |
a=-1.9376*lnx1+0.4753
(x1= Ultrasound power, [W/cm2]) |
b=-1.5277*lnx1+26.567
(X1=
Ultrasound power, [W/cm2]) |
Figure 5. Model function in relation to the real
values