Biomedical Engineering Reference
In-Depth Information
1991; Rahman I et al., 1996; Steinert M. et al., 1997; Gupte A.R. et al 2003; Bates T.C.,
Oliver J.D., 2004; Eschbach M. et al., 2004; Ganesan B. et al, 2007; Sachidanandham R., et
al. 2009). Possible presence of VBNC cells in environmental, clinical, food and other samples
makes results obtained with the use of only traditional culture techniques unreliable.
Lactococcus lactis
can enter VBNC state in stored cultured milk foods such as ageing
cheeses.
Detection of VBNC lactococci in the environment, milk products, organs of warm-
blooded organisms and biomedical preparations is of great microecological and biomedical
significance.
M
ATERIALS AND
M
ETHODS
For this study we chose 3 bacteriocin producing strains of
Lactococcus lactis
subsp.
lactis
(194, 729 and MGU). To induce VBNC state we used a synthetic medium that creates
carbohydrate starvation conditions, suggested by Ganesan B. et al, (2007). Formation of
VBNC cells was confirmed by difference between numbers of colony forming units
(CFU/ml) and total number of viable and dead cells. Percentage of viable cells was
determined in fluorescence microscope by their esterase activity after staining with Live/Dead
double cell staining kit, Fluka. Total counts were made in a Goryaev counting chamber.
CFU/ml value was determined by plate counts on the following medium (g/l):
sucrose - 20,
KH
2
PO
4
- 20, NaCl - 2, MgSO
4
- 0.2, yeast extract - 20, agar-agar - 15, pH 7.0.
R
ESULTS AND
D
ISCUSSION
Data on viability of
L. lactis
, obtained during six weeks of incubation in starved medium
are shown in the table. Our results show that after two weeks of incubation number of
CFU/ml dropped by 3 orders of magnitude (5х10
4
/ml average) and after 6 weeks - by 3-4
orders of magnitude (0,52 - 7,4х10
4
). Total viable counts remained fairly constant
(3,4х10
7
/ml average after 2 weeks and 6,79х10
7
after 6 weeks).
Culturable part of the population exhibited slowed growth rates: colonies reached
maximum size only by 48 hours. We found strain dependent differences in the rate of slowing
of colony growth speed. They were determined by comparing colony sizes after 24 hours of
culturing and times of their appearing (24-96 hrs.). At the beginning colonies reached 2-3 mm
in size within 24 hrs., after 2 weeks - 1-2 mm, and after 3 weeks colony size was less than 1
mm. Smallest colonies (after 24 hrs.) were observed for the strain 194. Their size varied from
barely visible to 0,5 mm. However upon the 4-th day the highest number of new colonies
(compared to the second day) was seen for strain MGU - up to 50% of total number.
Strain 729 showed phenotypic variability (emergence of transparent colorless colonies
with rough edges and less amounts of white exopolysaccharides). By the 5-th week their
numbers amounted to 93,6% (figure 1). This may be due to slowing of some metabolic
pathways in cells that are stored in carbohydrate starvation conditions for long periods. For
strain MGU in some cases we observed small colonies growing at the borders of bigger
colonies (figure 2). These colonies appeared several days after the big one reached its
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