BMOL30020 Practical 1

Name: Tony Corcoran

Student number: 0844443
Characterisation of the Biofilm forming capacity of Staphylococci epidermidis.
!ntroduction:
"im#:
Met$od#: "# %er manual&
'e#ult#:
()%eriment "& "##ay o* $aema++lutination by S& e%idermidi# #train# 1, 2 and 3:
Strain 1: Blood cells were diffuse, with no cells pelleting at the bottom of the plate.
Strain 2: Pellets were present at the bottom of the plate.
Strain 3: Some pelleting and diffuse blood cells.
()%eriment B& Con+o red a+ar te#t:
Strain 1 Blac!, dry"rough te#ture.
Strain 2 $ %ed, smooth te#ture.
Strain 3 $ %ed, smooth appearance yet stic!y to touch.
()%eriment C& -./ell %late ad$erence a##ay:
"ll ab#orbance read at a /a0elen+t$ o* 4-2nm&
1 2 7 8
S1 mean
Abs
492nm
Stressed S1
mean Abs
492nm
A 0.202 0.633 0.265 0.549 0.2335 0.591
B 0.209 0.493 0.289 0.398 0.249 0.4455
C 0.192 0.494 0.235 0.366 0.2135 0.43
D 0.226 0.431 0.279 0.259 0.2525 0.345
E 0.253 0.512 0.234 0.263 0.2435 0.3875
F 0.215 0.42 0.312 0.254 0.2635 0.337
G 0.153 0.651 0.245 0.426 0.199 0.5385
H 0.172 0.724 0.264 0.623 0.218 0.6735
Column 1 1 2 3 Strain 1 4 TSB
Column 2 1 8 3 Strain 1 4 TSB 4 45 (tO6
3 4 9 10 S2 mean Stressed S2
Abs
492nm
mean Abs
492nm
A 0.059 0.053 0.051 0.047 0.055 0.05
B 0.061 0.059 0.063 0.055 0.062 0.057
C 0.058 0.062 0.06 0.056 0.059 0.059
D 0.063 0.062 0.057 0.06 0.06 0.061
E 0.061 0.059 0.056 0.048 0.0585 0.0535
F 0.061 0.06 0.059 0.06 0.06 0.06
G 0.062 0.059 0.06 0.058 0.061 0.0585
H 0.049 0.05 0.055 0.05 0.052 0.05
Column 3 1 - 3 Strain 2 4 TSB
Column 4 1 10 3 Strain 2 4 TSB 4 45 (tO6
5 6 11 12
S3 mean
Abs
492nm
Stressed S3 mean
Abs
492nm
A 0.042 0.057 0.049 0.06 0.0455 0.0585
B 0.051 0.067 0.057 0.051 0.054 0.059
C 0.056 0.053 0.056 0.056 0.056 0.0545
D 0.059 0.062 0.052 0.078 0.0555 0.07
E 0.065 0.06 0.041 0.047 0.053 0.0535
F 0.056 0.06 0.043 0.06 0.0495 0.06
G 0.05 0.059 0.052 0.055 0.051 0.057
H 0.051 0.053 0.049 0.044 0.05 0.0485
Column 7 1 3 Strain 3 4 TSB
Column 11 1 12 3 Strain 3 4 TSB 4 45 (tO6
T$e#e re#ult# are *urt$er #im%li*ied:
Sample
Mean Abs
492nm
Stran 1 ! "SB 0.2340625
Stran 1 ! "SB ! 4
# Et$H 0.4685
Stran 2 ! "SB 0.0584375
Stran 2 ! "SB ! 4
# Et$H 0.056125
Stran 3 ! "SB 0.0518125
Stran 3 ! "SB ! 4
# Et$H 0.057625
8i#cu##ion:
()%eriment "& "##ay o* $aema++lutination by S& e%idermidi# #train# 1, 2 and 3:
Strain 1 shows the characteristics of a strong biofilm former in this e#periment. &#tensi'e
e#pression of the ica()BC operon under stress has led to high concentrations of P*(+ in the
sample. P*(+ is a stic!y polysaccharide, and pre'ents the hea'y red blood cells from pelleting at
the bottom of the plate. ,nstead, the red blood cells are loc!ed in their diffuse state, suspended in the
biofilm colony around them.
Strain 2did not e#press P*(+ when stressed. -he red blood cells in the sample pelleted at the
bottom of the plate.
Strain 3 displayed both pelleting and haemagglutination. -here is e'idence of P*(+ production,
but it is clear that the e#tent of this production is not sufficient to .hold/ the red blood cells. -he
results indicate that the strain may be a wea! biofilm producer, but we cannot rule out the
possibility that ethanol is an insufficient threat.
()%eriment B& Con+o red a+ar te#t:
-he results indicate that strain 1 is strongly Biofilm positi'e. -he colony was coloured blac! and
had a dry, stic!y te#ture. Strain 2, in contrast, was red in colour and had a smooth te#ture indicating
a Biofilm negati'e strain. Strain 3 was somewhat intermediate, being red and colour yet slightly
stic!ier than strain 2. -his indicates that strain 3 is a wea! Biofilm positi'e strain.
()%eriment C& -./ell %late ad$erence a##ay:
-he absorbance of strain 1 doubled when stressed. -his demonstrates the high capacity of strain 1 to
form biofilm, as red blood cells were trapped in a diffuse state
,n strain 2 samples, the absorbance remained unchanged under stressed conditions. -here is no
biofilm production, and so there is nothing to hold the red blood cells in suspension, thus they
pellet.
9ue#tion#:
:$at condition# in vivo mi+$t t$e bacteria e)%erience to %romote bio*ilm de0elo%ment;
&n'ironmental stresses pro'ided the e'olutionary pressure necessary for biofilm de'elopment to
arise in Staphylococci epidermidis. Such stresses in vivo could be heat shoc!, the presence of
antibiotics 0produced by other bacteria in the host, or administered to the host as therapy1or changes
in plasma p2.
()%lain /$y +ro/t$ o* bacteria in t$e %re#ence o* et$anol increa#e# P!"<PN"= %roduction
and bio*ilm de0elo%ment&
-he genes which encode P*(+ are located on the ica()BC operon. -he repressor for this operon
is encoded by the icaR gene. &thanol downregulates the e#pression of this gene, lea'ing the
ica()BC operon free for transcription, thus P*(+ is produced.
:$at ma>e# bacterial bio*ilm# re#i#tant to antibiotic# and $o#t immune re#%on#e#;
-he principle behind the protecti'e nature of the biofilm is altruism. Cells on the periphery of the
system are e#posed to attac! by the same stresses which were initially were present in the host.
2owe'er, the sheer number of cells pre'ents the threat from penetrating deep into the community of
bacteria. ,mmune responses launched against the biofilm colony will ha'e a dampened effect.
:$at *uture #trate+ie# mi+$t be u#ed to combat de0ice related in*ection#;
'e*erence#: