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Porównanie różnych metod pomiarowych charakterystyki wzrostu w wyborze pożytecznej flory mikrobiologicznej pochwy

E. Styková, H. Morvayová, F. Novotný, J. Pošivák, Ľ. Šťavová, I. Valocký

Klinika dla Koni, Uniwersytet Medycyny Weterynaryjnej, Koszyce, Słowacja

Streszczenie
W doświadczeniu tym porównano wyniki pomiaru charakterystyki wzrostu określonej trzema różnymi metodami.
Pomiar zmętnienia został wykonany na spektrofotometrze VIS Cintra 202 w zakresie częstotliwości 540 nm. Absorbancja mierzona była off line w próbkach o objętości 2 ml pobieranych z butelki fermentacyjnej o objętości 100 ml. Fermentacja przeprowadzana była w ruchu ciągłym na urządzeniu Roller Mixer SRT 9D. Próbki pobierane były co każde 2 godziny. W celu otrzymania odpowiedniej liczby wyników konieczne było wykonanie siedmiu 16-godzinnych eksperymentów prezentowanych w siedmiu 24-godzinnych eksperymentach. Wykonano 1 127 pomiarów na 22 próbkach.
Pomiar zmętnienia został wykonany na czytniku Synergy 4 Multi - Mode Microplate w zakresie częstotliwości 450, 540, 630 oraz 950 nm (NIR). Pomiary zostały wykonane przy pomocy płytek wiążących medium ze studzienkami Microtiter 96. Automatyczny pomiar wymagał dwóch 24-godzinnych cykli oraz dwóch płytek mikromiareczkujących. Pomiary zostały wykonane automatycznie i nie wymagały nadzoru personelu. Wytrząsanie i inkubacja były wykonywane automatycznie, bez przerywania. Indywidualne pomiary miały wyraźny, nie oscylujący trend, aż do momentu zakończenia wykładniczej fazy wzrostu, a odchylenia zdarzały się rzadko. Wykorzystanie różnych spektrów, w szczególności w zakresie NIR wymaga dalszych badań. W porównaniu z pomiarami off line w spektrofotometrze kuwetkowym, pomiary na czytniku Synergy 4 Multi - Mode Microplate mają wiele zalet z różnych punktów widzenia:

a. Możliwość porównania dużych ilości próbek w tym samym czasie
b. Zmniejszona jest ilość błędów subiektywnych
c. Zwiększona jest częstotliwość oraz dokładność pomiarów
d. Możliwość wykorzystania różnych spektrów
e. Znacząco zmniejszone jest zużycie próbek oraz odczynników
f. Znacznie zmniejsza się konieczność pracy personalnej

Krzywe otrzymane przy zastosowaniu technologii czytnika Synergy 4 Multi - Mode Microplate są wysoce powtarzalne i charakterystyczne dla poszczególnych szczepów.

Pomiar pH off line w interwale dwugodzinnym jest odpowiedni do stworzenia kryteriów krzywych wzrostu oraz produkcji metabolicznej. Potwierdzona została zależna od czasu interakcja pomiędzy pomiarem zmętnienia, a spadkiem pH.

Comparison of the different measuring methods of the growth characteristics in the selection of the beneficial vaginal microorganisms

E. Styková, H. Morvayová, F. Novotný, J. Pošivák, Ľ. Šťavová, I. Valocký

Horse Clinic, University of the Veterinary medicine, Košice, Slovakia

Abstract
In this method were compared the results of the growth characteristics measured by three different methods.
Measurement of the turbidity on the VIS spectrophotometer Cintra 202 in the spectrum sphere 540 nm. Absorbance was measured off line in the 2 ml samples taken from the 100 ml fermentation bottle. Fermentation was done during continuous motion on the Roller Mixer SRT 9D. Samples were taken every 2 hours. To obtain the necessary number of the results was needed to done seven 16 hours experiments presented in the seven 24 hour experiments. Were done 1 127 measurements on the 22 samples.
Turbidity measurement on the Synergy 4 Multi - Mode Microplate Reader in the spectrum spheres 450, 540, 630 and 950 nm (NIR). Measurements were done using the medium binding Microtiter 96 well plates. Automatic measurement required two 23 hours cycles and 2 micro titrate plates. Measurements were done automatically and didn´t require personal supervision. Shaking and incubation was done automatically, without interruption. Individual measurements had explicit, non - oscillating trend until the end of the exponential growth phase and deviations occurred rarely. Utilization of the different spectrums, particularly in the NIR sphere, needs further analysis. In comparison with the off line measurements on the cuvette spectrophotometer, measuring on the Synergy 4 Multi - Mode Microplate Reader has the advantages from the different points of view:

g. possibility to compare the large quantities of the samples in the same time
h. number of the subjective errors is decreased
i. frequency and accuracy of the measurements is increased
j. ability to utilise of the different spectrums
k. consumption of the samples and chemicals is significantly decreased
l. necessity of the personal work is considerably decreased

Curves obtained by the Synergy 4 Multi - Mode Microplate Reader technology are highly reproducible and characteristic for the particular strains.

pH off line measurement in the interval of the two hours is applicable for creating of the growth curves criteria and metabolite production. The interaction between the turbidity measurement and decrease in pH depending on time was confirmed.

Introduction
Uterine infections are one of the main causes of infertility in postpartum cows (R a j a l a- S c h u l t z, G r ö h n 1999). These infections increase the interval from calving to first recorded oestrous and the rate of services to conception. These variables reduce the reproductive performance in the dairy herd (L e w i s 1997), increase the costs and decrease the milk production. Antimicrobial treatment applied for metritis often lack efficacy due to microbial resistance and can leave residues in the milk, which must then be discarded (L o w d e r 1993). Such situations force producers to prefer to cull cows that would otherwise be productive and remain in the herd, before the therapy.
The use of probiotic products doesn´t cause the adverse effects and could prevent the undesirable consequences of the antibiotics.
Lactobacilli are the most frequently used productive microorganisms for the preparation of the probiotics. Lactobacilli, which are the part of the microflora in the genital tract, can help in maintaining its balance and stimulation of the immune mechanisms if are applied intravaginaly.
Acid production and pH influence
Lactic acid which is the characteristic fermentative product of lactobacilli, can reduce pH to the level in which the growth of the pathogenic flora is inhibited or destroyed. Low pH lead to decrease in the activity of metabolic enzymes and its denaturation. Only few bacterial strains are able to growth in pH lower than is the treshold for lactobacilli. However, pH measurement is not a reliable indicator of the growth or growth inhibition of bacteria. pH can give only a partial illustration of the concentration of the organic acids in the medium, because the large amount of these acids is non - dissociated. Bacterial wall cell is non - permeabile for dissociated acid fractions, whereas non - dissociative fractions are passively difunding through the membrane where are ionized depending on the intracellular pH. This leads to acidifying of the cytoplasm and production of the inhibitors (N e m c o v á, 1997).

Material and Methods

Sampling
Before taking samples, the external genitals were washed with hypermangan dilution. Samples were obtained by gentle swabbing of the vaginal wall and contamination was prevented by avoiding contact with the vulva. Vaginal swabs were taken with the carbon tampon Dispolab. After the arrival at the laboratory was each tampon put into the tube with 5 ml of PBS and vortexed for 30 s to suspend attached bacteria. Cultivation was done on the MRS agar. Samples were incubated in the thermostat during 48 hours at 39 °C. Cultivations were evaluated according to selection criterias. Colonies which grew on the solid medium were taken to the tubes with liquid LS broth. One colony was taken to the one tube. Colonies were cultivated for 24 hours at 39°C. Grow was judged by the increasing turbidity. From the colonies cultivated in the liquid medium was, after the vortexing, put 10 ml to MRS agar and spread by loop. Colonies were cultivated in the anaerostat during 48 hours at 39 °C. If on the MRS agar grew only one colony, this was used for the measurement of the growth curves. If on the MRS agar grew two or more colonies each of them was put to the LS broth and incubated in the anaerostat at 39 °C, until the pure colonies were obtained. For the turbidity measurement was 10 µl of the sample cultivated in the LS broth put to 50 ml of the LS broth and vortexed. Turbidity was measured in the cuvettes against distilled water on the Cintra 202 every 2 hours 16 x under the wavelength of 540nm.

pH measurement
The amount of the acids produced by lactobacilli was determined by pH measurement with pH meter every 2 hours 16 times.

Measurement of the turbidity on the Synergy 4 Multi - Mode Microplate reader
Measurements were done on the Synergy 4 Multi - Mode Microplate reader and medium binding 96 well Microtiter plates were used. Into the 1. column was put 300 µl of distilled water. Into the 2. column was put 300 µl of pure LS broth. Into the 3. column was put 290 µl of the LS broth and 10 µl of the broth with multiplied lactobacilli strains. Measurings were done every 15 min during the two 23 hours cycles under the different wavelengths: 450 nm, 540 nm, 630 nm and 950 nm (NIR).
Another selection criterium was the growth of the selected strains under the aerobic conditions on the blood agar ( Imuna Pharm, a.s., Šarišské Michaľany, SR). From all samples in which the turbidity was measured, was in the same time put 10 µl on the blood agar containing the ram blood. Samples were cultivated for 24 hours at 39 °C. Samples with fast and expansive growth under the aerobic conditions were removed.
Selected strains were further selected by microscopical morphology, magnification 1: 1 000.

Results

Graph 1. Mean values from  measurements at 540 nm measured on the Cintra 202  (3 repeatings)

 


Graph 2. Mean values of pH measured on the pH meter

 

 
Graph 3. Mean values from 8 repeatings at 450 nm measured on the Synergy 4 Multi - Mode Microplate reader   Graph 4. Mean values from 8 repeatings at 950 nm measured on the Synergy 4 Multi - Mode Microplate reader

 


Discussion
The goal of this experiment was to compare the quality of the measurements focused on the evaluation of the growth characteristics of the selected bacterial strains, which grew on the MRS agar under anaerobic conditions.
Off line spectrophotometric measurements in the cuvettes on the Cintra 202 can be used, but the growth is considerably influenced by the interruptions during the preparation of the samples for the measurements every 2 hours. These measurements are demanding in time and labour work.
Synergy 4 Multi - Mode Microplate reader is using mounted shaking and thermostatic unit, so the measurements can be done more frequently and at the various spectrums. Measurements were done every 15 minutes during the 23 hour period.
The absorbance at 450nm, 540 nm, 630 nm and 950 nm was tested.
In comparison of the suitability of the spectrums was the turbidity shape of the curves at 450 nm, 540 nm and 630 nm to a large extent equal with the absolute drift in the measured turbidity values. Higher turbidity values were measured at the lower wavelengths (Graph 3).
Shape of the curves measured at 950 nm was similar, but at the absolute values of the absorbance was displayed the subtraction of the absorbance of the plastic cuvette material and water from the Blank sample. Water and plastic cuvette material are highly absorbable in the NIR spectrum and for this reason are in the curves shapes also negative values (Graph 4).
Absorbance values had in the individual measurements minimal variance. Contamination was displayed in 15 – 20 % of the repeatings in the individual strains. Contamination was absolutely obvious from shape of the growth curve. Curves which displayed explicit marks of contamination were not evaluated.
From the graphs is evident, that the growth curves of the 22 strains can be divided into 2 groups of 6 – 8 strains. These strains have similar growth characteristics.
After the 12 hour measurements some curves have slightly saw – tooth shape, which is probably caused by the aggregation of bacteria to larger aggregates. This phenomenon can be observed in some strains during the subjective growth evaluation in the tubes.
Growth curves based on the pH measurement are obviously divided in 2 groups, but these groups are not correlating with the groups divided due to turbidity measurement.

Conclusion
Measurement of the growth curves on the Synergy 4 Multi - Mode Microplate reader very effectively and exactly complete the growth evaluation on the selective liquid mediums. This method will be standardly utilize for the typification of the beneficial microorganisms together with the utilization of the selective mediums.


Acknowledgements
This study was supported by a project from
1. the Research and Development Support Agency APVV-20-062505.
2. the Research and Development Support Agency AV 4/0009/07

University of the Veterinary Medicine, Komenského 73, 041 81 Košice, Slovak Republic


References
1. LEWIS, G. S. (1997) Symposium: health problems of the postpartum cow. J Dairy Sci 80, 984 - 994
2. LOWDER, M. Q. (1993) Diagnosing and treating bovine postpartum endometritis. Vet Met 88, 474
3. NEMCOVÁ, R. (1997) Vet Med - Czech, 42, 19- 27
4. RAJALA - SCHULTZ, P. J.; GRÖHN, Y. T. (1999) Culling of dairy cows. Part I. Effect of diseases on culling in Finnish Ayrshire cows. Prev Vet Med 41, 195 - 208





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