ISOLATES 3.1 AIMS
3.3 MATERIALS AND METHODS
3.3.1 Culture Methods
A pure culture of each of the bacteria Isolated from Tocil Lake
(Chapter 2), was inoculated into the following media:
(b) Glucose medium containing 0.544g KH2P04, (Fisons, Loughborough!,
0.38g NHaC/2 (Fisons, Loughborough), 0.6ml of a salt solution consisting
of 10gl“ ' MgS0.».7H20 (Fisons, Loughborough), 0.4gl_1 FeSCLt.THzO (Fisons,
Loughborough) and 0.2g glucose (Fisons , Loughborough). The glucose and
FlLtC^ were prepared in 10ml volumes of distilled water and autoclaved
separately at 121°C for 10 mins and added aseptlcally to the KH2PO.4
which had been sterilised by autoclaving at 121°C for 15 mins. The salt
solution was prepared in 100ml volumes in distilled water, filter
sterilised (0.45 p cellulose acetate filter) and added to the medium to
give a final volume of 100ml.
(c) A mannose medium prepared as (b) but with 0.2g mannose (Fisons ,
Loughborough) Instead of glucose.
(d) A sucrose medium prepared as (b> but with 0.2g sucrose (Fisons ,
Loughborough), Instead of glucose.
The inoculated flasks were Incubated at 15°C in a shaking incubator for
16 hrs. These flasks were used as stock cultures and stored at 4°C for
up to two weeks.
3.3.2 Glass Coversllps
The surfaces used in these experiments were 16mm diameter glass
coversllps (Chance Propper Ltd, Warley, England). The coverslips were
washed in a mixture of concentrated nitric acid and concentrated
sulphuric acid (1/1) for 48 hrs and then rinsed with sterile distilled
water six times. After drying, the coversllps were placed in sections of
sterile silicon tubing (Vatson and Marlow, England) 15mm in diameter and
16mm in length. This silicon tubing enabled the coversllps to be handled
3.3.3 Absorbance versus Viable Counts
The relationship between absorbance and viable count for each
bacterium in Section 3.3.1 was determined in the various media used in
this section to aid in the estimations of bacteria present in a flask
during attachment experiments. The relationships between absorbance and
viable counts of these bacteria in nutrient broth were determined by
inoculating 1ml of each stock solution Into 100ml of nutrient broth.
These cultures were then incubated at 15°C in a shaking incubator at 150
rpm. Growth was followed initially turbidometrically at 540 nm using a
Corning colorimeter 252 and by doing viable counts before each
colorimeter reading. An identical procedure was used to determine the
growth rates of each bacterium used in attachment experiments on each of
the media used.
3.3.4 Attachment Experiments
Flasks were set up as In section 3.3.1 and 1ml of a stock culture
was aseptlcally inoculated into each flask. These flasks were Incubated
at 15°C until late stationary phase was reached. Four coverslips were
aseptically added to each flask and the flask was Incubated in a shaking
incubator for 2 hrs at 150 rpm. After incubation, the coverslips were
removed and rinsed in sterile 0.1M phosphate buffer (pH 7.4) or sterile
nutrient broth to remove any loosely attached bacteria. The coverslips
were placed in Bouln's fixative for 30 secs, rinsed in sterile distilled
water and stained with crystal violet (0.5% w/v> (Flsons, Loughborough).
The coverslips were rinsed once more in sterile distilled water and left
to dry at room temperature. The coverslips were mounted on glass
microscope slides and examined under oil immersion using a light
The bacterial population remaining In the flask was determined by
doing a viable count on nutrient agar plates.
3.3.5 Detachment
Sometimes It was Impossible to count the bacteria on a surface due
to the presence of large numbers of cells on the surface. Also If there
was more than one cell type present, these could not always be
distinguished accurately and therefore a microscopic count of the
different bacteria present could not always be obtained. In such cases,
the bacteria were counted by removal from the surface. To remove the
cells, surfactants were used which have been shown to remove bacteria
from surfaces under certain conditions (Ball, 1986). The surfactants
used were BGTA (Ethylene Glycol-bis (p-aminoethyl Ether) N,K,N, H-Tetra-
acetic Acid) (Flsons, Loughborough) and Tween-20 (Flsons, Loughborough).
These surfactants were first tested on blofllms prepared using bacteria
selected for future attachment experiments to determine their efficiency
at removing these bacteria from surfaces.
Once attachment experiments had been performed on glucose media
(Section 3.3.4) using selected Gram-negative and Gram-positive bacteria,
countable blofllms were obtained. These biofilms were then subjected to
various experimental procedures to test which surfactant was best suited
for the detachment of these bacteria from the surfaces. After the
surfaces were washed to remove loosely attached cells, the surfaces were
placed In unlversals containing 200, 100, 50, and 25 ppm Tween 20 or 20,
10, 5, and lmg EGTA in a total volume of 5ml sterile distilled water.
These unlversals were Incubated at 15°C or 37°C for 30, 60, and 90 mins
In shaking Incubators at 50, 100, 150, and 200 rpm. The number of
were fixed and stained to determine the number of cells still on the
surface (Section 3.3.4). During the detachment process, bacterial cells
sometimes became non-viable and this had to be accounted for when the
detachment percentages were determined.
Once the best detachment method was obtained the process was
performed on each bacterial Isolate selected for future attachment
experiments, to assess the percentage of detachment under these
conditions. Detachment experiments were performed on blofilms grown on
nutrient broth and glucose medium (Section 3.3.1) to assess If the
nutrient conditions affected the detachment of the bacteria.
3.3.6 The Effects of Subculture on Bacterial Attachment
The bacteria Isolated In Section 2.3.1 were subcultured on nutrient
agar plates and glucose agar plates (Section 2.3.3) and Incubated at
15°C. Stock cultures of each bacteria were prepared (Section 3.3.1) and
attachment experiments were performed as In section 3.3.4 using glucose
medium and nutrient broth. This procedure was repeated six times to
determine the attachment abilities of each Isolate after each
subculture.
The results obtained from the subculture attachment experiments were
expressed as a proportion of the total bacteria attached against the
total bacteria present in the flask,
The number of fields of view present during the attachment experiments
with four surfaces were 63206 obtained by determining the area of the
microscopic field of view (ITr2 ), dividing this number into the area of
sides to the glass coverslips. The results from future attachment
experiments were also expressed In this form.
3.3.7 The Bffect of Culture Age on Bacterial attachment
Bacterial Isolates not selected for future experiments (Section
3.3.4) were inoculated Into nutrient broth (Section 2.3.1) and glucose
medium (Section 3.3.1). These flasks were Incubated at 15°C until
stationary phase was reached In an orbital shaker Incubator and stored
at 4°C. Nutrient agar and glucose agar plates (Section 2.3.3) were
streak Inoculated with these bacteria and these plates were Incubated at
15°C in a standing incubator until full growth was obtained.
Attachment experiments were performed on the bacteria from each of
the storage media (Seclton 3.3.4), in the cases of the solid medium the
isolates were first grown up in the corresponding liquid medium to
enable the attachment experiments to take place. The bacterial
population In each flask was estimated by viable count, and these
experiments were repeated every seven days.
3.3.8 The Bffect of Bacterial Concentration on Attachment
The bacteria selected in Section 3.3.4 were inoculated into 10
nutrient broth flasks and 10 glucose medium flasks (Section 3.3.1). The
flasks were Incubated at 15°C until stationary phase was reached. The
bacteria in the flasks were diluted with sterile nutrient broth or
sterile glucose medium to obtain flasks containing different bacterial
concentrations in 100ml. Attachment experiments were performed on each
flask (Section 3.3.4) and the concentration of bacteria present in each
flask was estimated by viable count. These experiments were performed to
3.4 RESULTS
3.4.1 Attachment Abilities of Isolates
The results obtained from Initial attachment experiments (Table 3. 1)
Indicated no relationship between the attachment of Gram-negative and
Gram-positive bacteria, e.g. all the bacteria attached differently to
the glass coverslips. There was no relationship between generic
classification and extent of their attachment, e.g. Pseudomonas (1)
attaches differently from Pseudomnnas (3>. Similar results have been
reported in the literature (Caldwell, 1986; Hsieh, 1986; Zvyagintsev,
J
1979).
There were variations in bacterial morphology observed, e.g.
Flavobacterlum became elongated in nutrient broth medium and the
Bacillus species was reduced In size In the glucose medium. These
phenonemon have been reported in other studies (Caldwell, 1986;
Marshall, 1980). These results were considered when selecting the
bacteria to be used In the subsequent attachment experiments. The object
was to obtain bacteria which varied in their ability to attach to the
glass coverslips under different nutrient conditions. This was an
Important bacterial trait to be Investigated in subsequent attachment
experiments. Bacteria were not selected if they changed morphology
during attachment experiments as this would have complicated their
identification on attachment.
3.4.2 Detachment
The results obtained from the numerous detachment experiments
(Appendix Tables 2.1-2.24) indicate that the best detachment of the
Gram-negative and Gram-positive bacteria was obtained with Tween 20 at
TABLE 3.1 Attachment abilities of the bacterial Isolates under different nutrient conditions.
BACTERIA NUTRIENT