ORAL BIOFILMS

Most of the time in the microbiology lab, we study free-floating bacteria in broths or bacteria incolony forms, and generally in pure culture. However, in the real world bacteria are usuallyinteracting with other species in pretty sophisticated ecosystems. This assemblage of variousorganisms attached to a surface is called a biofilm , and the organisms that comprise it caninclude bacteria, plants, fungi, protozoa, and even multicellular animals, depending on where thebiofilm is.

These biofilm communities attach to teeth, to the inside of a toilet, on a sponge, in a catheter,inside of a blood vessel, in the middle ear, on contact lenses, metal pipes, and so on. Interactingmicroorganisms act differently than if the microbes were living independent to eachother. Different chemical by-products are produced and the surfaces that they grow on can bedamaged by these physical and chemical interactions among the various species. In addition,interacting species can produce infections that are difficult to eliminate, possibly because thephysical architecture of the biofilm protects the individual organisms from antibiotics (likeschooling fish are protected by moving closely around together) or because there is no ONEmicrobe causing the infection. In the body, the biofilm community can produce inflammation anddiscomfort.

"Biofilm formation Sequence" © Mark Wiencek, author. Licensed for use, ASM MicrobeLibrary.

In this lab exercise, we are interested in oral biofilms---on teeth and on toothbrushes. Plaque isa protective layer that the bacteria live within, and, in fact, produce: It consists of bacteria, and theslime layers that they have around their cells and food particles. Within the plaque they areprotected, and they are also free to metabolize and produce erosive by-products which thencause tooth decay, destroying the dentin of the teeth. If the plaque is not eliminated, becoming

You will need to bring a sample of plaque, as well as a used toothbrush. The plaqueshould be scraped off into a plastic boat (take one home from lab) first thing in themorning, BEFORE brushing your teeth. The toothbrush has to have been used for acouple of months, but don't treat it any differently than normal.

harder and denser, it causes the gum to recede and the tooth can lose attachment to thejawbone. This is periodontal disease.

In the procedure below, you may notice the specimens are not as diluted for the toothbrush asthose for plaque, mainly because some oral microorganism will not live as long on thebrush. Also note that for some of the media, the sample being spread on the plate is not verydilute. That is because we are using some very selective media that will inhibit most of thebacteria.

Mitis-salivarious agar for isolation of oral Streptococcus species Mannitol salt agar for isolation of halophiles like Staphylococcus and some of the Strep

sepcies MacConkey’s agar for the gram – bacilli like E. coli

The Biofilms Hypertextbook - http://www.cs.montana.edu/ross/personal/intro-biofilms-s1.htm

OBJECTIVES:

Analyze biofilms of the mouth.Compare bacteria in plaque and on a toothbrush.Identify some of the common bacteria in this biofilm community.

MATERIALS NEEDED: per table

a regular toothbrush, used for at least 2 monthsplaque scraped from teeth first thing in morning, before brushing teethsonicatorplastic toothpickssterile mortar and pestle2 glass hockey sticks (for use in spread plates.)ethanol containers for flaming glass sticksbottle of phosphate buffer solution (~125ml)9 sterile tubes2 - 10ml pipettes10 - 1ml pipettes2 sizes of pi-pumps10 blood agar (or BHI may be used)2 mannitol salt agar2 MacConkey's agar (in Photo Atlas)2 Mitis-salivarius agar platetriple-beam balance

Center for Biofilm Engineering - http://www.biofilm.montana.edu/

THE PROCEDURES:

Dispense 9ml of phosphate buffer solution into 9 sterile tubes.5 will be used for the plaque dilutions, 4 for the toothbrush dilutions.

PLAQUE

1. Using a toothpick, remove as much plaque as possible from between your molars. Thesample will be placed into a weigh boat (be sure to get it off of the toothpick).

2. Make a 1/10 dilution of the plaque in phosphate buffer solution by adding 10ml of the diluentto the weight boat and mixing it well .

3. Pour the 1/10 dilution + plaque into a mortar and pestle and crush well to make an evensuspension.

4. Make 10 fold dilutions by using 9ml phosphate buffer solution dilutions. Be SURE to mixeach dilution well, and use new pipettes.

o Transfer 1ml of the 1/10 into 9ml phosphate buffer, using a fresh pipette: This is a 10 -2 .o Transfer 1ml of the 10-2 into 9ml phosphate buffer, using a fresh pipette: This is a 10-3.o Transfer 1ml of the 10-3 into 9ml phosphate buffer, using a fresh pipette: This is a 10-4.o Transfer 1ml of the 10-4 into 9ml phosphate buffer, using a fresh pipette: This is a 10-5.o Transfer 1ml of the 10-5 into 9ml phosphate buffer, using a fresh pipette: This is a 10-6.

5. Plate out 0.1 ml aliquots using the spread plate method, using alcohol-flamed glass hockeysticks.

o Make blood plates (or BHI agar plates) from the dilutions labelled 10-2, 10-3, 10-4 , 10-5,and 10-6, as shown in the diagram below.

o Make mannitol salt agar plates from the 1/10 dilutions.o Make MacConkey's plates from the 1/10 dilution.o Make Mitis-salivarius plates from the 1/10 dilution.

TOOTHBRUSH

1. Cut the top off of the toothbrush with a pair of pliers and cutters or diagonal cutters, allowing itto drop into the glass container. Dispense 10ml of phosphate buffer solution into the bottlewith the toothbrush head. This is your 1/10 dilution.

2. The sonicator is filled 2/3 with water, so place the bottle into the sonicator, and turn on for 30seconds. Be sure to hold the bottle sonicator while it is sonicating.

3. Make 10 fold dilutions by using 9ml phosphate buffer solution dilutions. Be SURE to mixeach dilution well, and use new pipettes.

o Transfer 1ml of the 1/10 into 9ml phosphate buffer, using a fresh pipette: This is a 10 -2 .o Transfer 1ml of the 10-2 into 9ml phosphate buffer, using a fresh pipette: This is a 10-3.o Transfer 1ml of the 10-3 into 9ml phosphate buffer, using a fresh pipette: This is a 10-4.

o Transfer 1ml of the 10-4 into 9ml phosphate buffer, using a fresh pipette: This is a 10-5.4. Plate out 0.1 ml aliquots using the spread plate method (use alcohol-flamed hockey sticks).

o Make blood plates (or BHI agar plates) from the dilutions labelled 1/10, 10-2, 10-3, 10 -4 ,and 10-5, as shown in the diagram below.

o Make mannitol salt agar plates from the 1/10 dilution.o Make MacConkey's plates from the 1/10 dilution.o Make Mitis-salivarius plates from the 1/10 dilution.

Incubate all plaque plates and toothbrush plates in 37 C incubator as follows. all BAP (or BHI) and Mitis-salivarius plates in candle jars mannitol salt in ambient air incubator MacConkey's in ambient air incubator

INTERPRETATION:

1. Remove all plates from the incubators, and separate them into 2 groups---plaque ortoothbrush.

2. Determine the number of bacteria per plaque sample. This will not be per gram since youhad much less than a gram sample of plaque.

3. Determine the number of bacteria per toothbrush head, using counts from blood or BHI.4. Analyze each type of medium for bacteria for BOTH toothbrush and plaque:

BAP or BHI - for total count of facultatives and aerobes Mannitol salt - for salt-resistant Staphylococcus species, Staph aureus turns

yellow (see exercise in lab manual) MacConkey's - for enteric gram - rods, coliforms turn red and non-coliforms are

clearish Mitis-salivarius agar - small blue colonies (Strep mitis), blue gumdrop-like colonies

(Strep salivarius) and dark blue/black, shiny colonies (Enterococcus faecalis)5. Determine the number of bacteria per toothbrush head for each group of bacteria, based

on each individual media type.6. Gram stain at least one colony on each type of medium and record the gram reaction,

arrangement, and shape.7. Report results last day of lab.

Data presentation and various bacterial countsWere your dilutions adequate for the bacterial counts on the different media?How did the counts for plaque vary from those of the toothbrushes?

credit to Dr JOANNA VERRANManchester Metropolitan University

QUESTIONS:

1. What is the purpose of each kind of medium?

2. Would you expect more or less anaerobes than aerobes? WHY?

3. What is the significance of biofilm presence in medical equipment?

Fall 2011 - Jackie Reynolds, Richland College, BIOL 2421