Medical Oral Microbiology Topic A - 12
Name: Date:
A12, p. 1
Oral microbiology III - biofilm
Task 1: Microscopy of dental plaque
Using and sterile stick, get dental plaque. Make
smear and Gram stain. Describe and draw the
objects. Mention clusters of bacteria, especially their
shape and colour.
Task 2: Effect of teeth cleaning to oral biofilm
Wash your mouth
by a solution of
given stain
according to
teacher´s
instructions and
observe. Stained
places are covered
by biofilm. Describe places, where biofilm is the most
denese.
Result: Biofilm was mostly present at following
places: ______________________________________
____________________________________________
Task 3: Diagnostics of microbes colonizing catheters
a) Qualitative method multiplication in broth
Extracted central venous catheter (CVK) was put into cultivation medium and cultured 24 hodin. After that, the
turbid cultivation médium was inoculated onto blood agar. Assess growth of microorganisms onto blood agar.
b) Semi-quantitative method (Maki method)
Extracted CVK was rolled on the surface of the blood agar, which was cultured after that. Evaluate growth
microorganisms and count grown colonies. As significant take amout of colonies >15, less than 15 colonies
should be consedered to be contamination. If there are clearly more than 100 colonies, do not count them and
write down simply ,,> 100".
c) Quantification acording to catheter sonification
Extracted CVK is put into 10 ml of saline and after that ultrasound effects on it, destroying the biofilm structure
and releasing individual bacterial cells. 100 microlitrs of such suspension should be inoculated directly onto
blood agar and diluted by a sterile loop onto the whole agar surface. According to teacher's instructions, perform
sonification of catheter. Inoculated blood agars place into the termostat to 37 °C.
Onto prepared Petri dish, count how many colonies grew onto blood agar and count the number of bacteria
adhering onto the cathether surface. If there are clearly more than 100 colonies, do not count them and write
down simply ,,> 100"
Medical Oral Microbiology Topic A - 12
Name: Date:
A12, p. 2
Results:
3a 3b 3c
Estimated number of
organisms
Which of the methods enables to detect and to quantify not only bacteria present onto the surface of the catheter,
but also in its lumen? _________________________________________________________________________
What methods enable us to quantify the amount of bacteria adhering to the catheter surface?
__________________________________________________________________________________________
What is the sense of quantification of a microbe izolated from a catheter? _______________________________
Task 4: Influence of saccharides concentration on biofilm growth
dynamics
Into individual wells of a microtitration plate with BHI medium supplemented by 0 %, 2 %, 4 %, 8 % of glucose,
Streptococcus mutans strain was inoculate. After 2, 8, 16, 24 hrs of culture at 37 °C the well were three times
washed. The biofilm layer, strongly adhered onto the surface was stained by 20 minute action of gentiane violet.
The remaining dye was removed from wells by a careful washing. Intensity of colour of wells is measured by a
spectrophotometer and correspond to the thickness of the biofilm layer.
On a sheet of paper you have results of spectrophotometric measurment of intensity of well colours. From given
result, draw a graph of dynamics of biofilm formation in correlation to glucose concentration and time. (For each
time and concentration, six wells are measured; choose always an approximated average, it is not necessary to
count very preciselly.)
Average
values*
2 h 8 h 16 h 24 h
0 %
2 %
4 %
8 %
*absorbance values, approx. average of all
six wells that were kept at the same glucose
concentration an the same time
Medical Oral Microbiology Topic A - 12
Name: Date:
A12, p. 3
Check up questions:
1. What is biofilm?
2. What are the main components of the biofilm?
3. What is the importance of oral biofilm with relation to dental caries?
4. What more diseases are influenced by a biofilm?
5. What complicates treatment of biofilm infections?