Posted: September 13th, 2017

Microbiology – Biofilm

Paper, Order, or Assignment Requirements

 

Please write an introduction (350 words), material and methods, results and a conclusion.

 

Week 10

Introduction: 350 Words about biofilms and include: (aims of the experiment: 1- Using a sample of 96-well microliter plate assay to compare the ability of a range of bacterial species and strains to form a biofilm by choosing from a bacterial species; Streptococcus oralis and Streptococcus infantis to form a biofilm.

 

Materials:  

• Bacterial cultures (Streptococcus oralis and Streptococcus infantis)
• 96 well microtitre plate.
• Empty, sterile glass universals.(3)
• Sterial BHI (broth 100ml)
• Gilson pipetman ( 1x P200; 1xP1000).
• Sterile Tips.

 

 

• Experimental design:

You will be growing biofilms in plastic 96-well plates. You will inoculate each well with a given strain or mixture of strains and allow biofilms to develop for 1 week. Next week, you will quantify the relative levels of biofilms formation using a crystal violet staining method.

Hypothesis made: There will be a synergistic interaction between streptococcus oralis and streptococcus.

96-well plate

• 1, 2 and 3 contained 50 µl of Streptococcus oralis each.
• 1, 2 and 3 contained 50 µl of Streptococcus infantis each.
• 1, 2 and 3 contained 50 µl of Streptococcus oralis and 50 µl of Streptococcus infantis.
• 1, 2 and 3 contained 50 µl of Brain Heart Infusion broth.
• Setting up the biofilms
• To prepare bacterial starter cultures, find the bacterial cultures that you need to set up your experiment. Each broth culture has been grown overnight in Brain Heart Infusion broth, a rich culture media. The optical density of the culture is OD₆ₒₒ1.0

Optical density is a measure of turbidity, or bacterial cell density. We can assume that there are roughly 1 x 10⁹cfu/ml in a culture of OD₆ₒₒ 1.0

Calculate the dilution factor needed to creat a starting culture with (optical density 0.05) 5 x 10⁷ cfu/ml

C1 X V1 = C2 X V2

C= Concentration (number of cells)

V= Volume (calculated in step 1.5 + 1 ml to ensure you have enough)

• Set up each starter culture in a sterile glass universal bottle using sterile BHI broth as the diluent and clearly label (3 bottles in total; strain 1; strain 2; and BHI only).
• Aseptically add the cultures to your microtitre plate as detailed on your plan.
• Put in the tray for incubation at 37 degrees for 7 days.

 

Week 11

• Quantifying biofilm production using Crystal Violet

Materials

• 96 well microtitre plate ( from the previous week)
• Plate reader (1 in lab)
• Pasteur pipettes
• 250 ml Duran Flask labelled waste
• Sterile PBS (100 ml)
• Gilson pipetman (1xp200)
• Sterile Tips (1x box 200 µl)
• Drying incubator (37 degrees)
• 1% Crystal Violet (30 ml)
• 100 % Ethanol (20 ml)

 

3.1 Recover your microtitre plate from last week and take it to the plate reader to read the optical density (wave length 600 nm) of each well. This will give you an indication of how well you cultures have grown relatives to one another. Record results in a table:

 

 

 

Value	1	2	3	4	5	6	7	8	9	11	12
A	2.9624	3.9269	2.3308	0.0481	0.0472	0.0553	0.0474	0.0477	0.0489	0.0477	0.0473
B	0.0654	0.2218	0.0477	0.0535	0.0451	0.0476	0.0473	0.0477	0.0478	0.0473	0.0517
C	3.1331	2.2847	5.0103	0.0481	0.0477	0.0504	0.0484	0.0485	0.0481	0.0513	0.0512
D	0.0479	0.0488	0.0493	0.0482	0.0515	0.0477	0.0481	0.0500	0.0489	0.0503	0.0490
E	2.9001	2.3915	2.3141	0.0481	0.0475	0.0530	0.0489	0.0457	0.0516	0.0524	0.0532
F	0.0505	0.0505	0.0474	0.0477	0.0479	0.0477	0.0478	0.0486	0.0477	0.0488	0.0488
G	2.1108	1.4521	1.6244	0.0496	0.0509	0.0506	0.0501	0.0482	0.0502	0.0554	0.0494
H	0.0475	0.0479	0.0492	0.0475	0.0525	0.0488	0.0484	0.0474	0.0476	0.0510	0.0476

 

 

10
0.0488
0.0484
0.0499
0.0479
0.0479
0.0495
0.0496
0.0482

 

 

 

 

3.2 Carefully remove the liquid (planktonic) phase of each well and dispose all into a 250 ml glass Duran flask marked BIOLOGYCAL WASTE.

3.3 Fill each well with sterile PBS using a Pasteur pipette.

3.4 Carefully remove the PBS from each well using a Gilson pipette and dispose of all liquid waste into the 250 ml glass Duran flask marked BIOLOGICAL WASTE.

3.5 Repeat steps 3.3 and 3.4. These steps will remove all planktonic cells, and those that are just sitting on the plastic surface, but will not disturb the biofilm.

3.6 Remove as much liquid as possible then transfer the plate to the 37 degrees  incubator to air dry (lid off) for 15 mins.

3.7 Once the plate is dry add 100 µl of 1% Crystal Violet to each well and incubate on the bench for 15 mins.

3.8 Carefully remove the crystal violet stain and dispose of all waste into 250 ml glass Duran flask marked BIOLOGICAL WASTE.

3.9 To remove unbound stain repeat steps 3.3 and 3.6

3.10 Once the plate is dry add 100 µl of 100% ethanol to each well to re-solubilise the bound crystal violet stain.

3.11 Replace the lid on your plate and take it to the plate reader to read the optical density (wavelength 570 nm) of each well. This will give you a semi-quantitative measurement of biofilm density. Record the results:

Sample

1

2

3

4

5

6

7

8

9

10

11

12

A

Un_0001 1/1

Un_0009 1/1

Un_0017 1/1

Un_0025 1/1

Un_0033 1/1

Un_0041 1/1

Un_0049 1/1

Un_0057 1/1

Un_0065 1/1

B

Un_0002 1/1

Un_0010 1/1

Un_0018 1/1

Un_0026 1/1

Un_0034 1/1

Un_0042 1/1

Un_0050 1/1

Un_0058 1/1

Un_0066 1/1

C

Un_0003 1/1

Un_0011 1/1

Un_0019 1/1

Un_0027 1/1

Un_0035 1/1

Un_0043 1/1

Un_0051 1/1

Un_0059 1/1

Un_0067 1/1

D

Un_0004 1/1

Un_0012 1/1

Un_0020 1/1

Un_0028 1/1

Un_0036 1/1

Un_0044 1/1

Un_0052 1/1

Un_0060 1/1

Un_0068 1/1

E

Un_0005 1/1

Un_0013 1/1

Un_0021 1/1

Un_0029 1/1

Un_0037 1/1

Un_0045 1/1

Un_0053 1/1

Un_0061 1/1

Un_0069 1/1

F

Un_0006 1/1

Un_0014 1/1

Un_0022 1/1

Un_0030 1/1

Un_0038 1/1

Un_0046 1/1

Un_0054 1/1

Un_0062 1/1

Un_0070 1/1

G

Un_0007 1/1

Un_0015 1/1

Un_0023 1/1

Un_0031 1/1

Un_0039 1/1

Un_0047 1/1

Un_0055 1/1

Un_0063 1/1

Un_0071 1/1

H

Un_0008 1/1

Un_0016 1/1

Un_0024 1/1

Un_0032 1/1

Un_0040 1/1

Un_0048 1/1

Un_0056 1/1

Un_0064 1/1

Un_0072 1/1

10	11	12
10	11	12
Un_0073 1/1	Un_0081 1/1	Un_0089 1/1
Un_0074 1/1	Un_0082 1/1	Un_0090 1/1
Un_0075 1/1	Un_0083 1/1	Un_0091 1/1
Un_0076 1/1	Un_0084 1/1	Un_0092 1/1
Un_0077 1/1	Un_0085 1/1	Un_0093 1/1
Un_0078 1/1	Un_0086 1/1	Un_0094 1/1
Un_0079 1/1	Un_0087 1/1	Un_0095 1/1
Un_0080 1/1	Un_0088 1/1	Un_0096 1/1

 

 

 

For conclusion please compare the biofilm density of each strain or mixture of strains and if the hypothesis was correct?