Lawn Plate Preparation:

• Wearing gloves, students should clean the bench surface with Virkon Disinfectant, drying with paper towels.

• Students set up and light a Bunsen Burner so that the yellow safety flame is in operation. They place the disinfected laminated sheet on the bench in front of the Bunsen Burner – this is now the students sterile working surface.

• Students then place the following equipment onto their laminated sheet:

  ·  Bottle of Bacterial Broth Culture

  ·  Sterile agar plate

  ·  Sterile Spreader

  ·  Sterile 1ml Syringe

  ·  Forceps

  ·  Filter Paper Discs

  ·  Discard Pot containing Virkon Disinfectant

• Students also need access to a range of antimicrobial substances, a marker pen, scissors and sticky tape.

• On the underside of the agar plate provided, students should label the plate sing the marker pen with their initials, class, date and name of the bacterial culture (E-Coli K12).  The plate is divided into three sections and each is labelled with the antibiotic to be tested (if using Antibiotic Mast Rings, the plate does not need to be divided into sections).

• The Bunsen Burner should now be switched to the roaring blue flame.  This creates an updraught around the students’ working area.  Students should now thoroughly wash their hands using hot water and liquid handwash/soap.  They should dry their hands using paper towel.

• Next, working as closely to the Bunsen Burner as is safe and possible, they should unwrap their sterile syringe and hold in one hand.

• Their other hand should pick up the McCartney Bottle containing the Bacterial broth, removing the lid, but without putting the lid down onto the working surface.  The open neck of the McCartney Bottle is then gently wafted through the blue Bunsen flame.

• The sterile 1ml syringe is now inserted into the bacterial culture bottle and 0.5ml of the bacterial broth is drawn up into the syringe.

• Without putting anything down on the bench surface, the open neck of the McCartney Bottle is again gently wafted through the blue Bunsen flame and the lid is replaced.

• Students now need to open their agar plate slightly, just enough to insert the mouth of the syringe and the bacterial broth is ejected from the syringe onto the surface of the agar.  The syringe is removed, the agar plate lid is closed and the used syringe can now be placed into the discard pot containing Virkon Disinfectant, ensuring that the disinfectant is drawn in to fill the syringe.

• The agar plate lid again is opened enough for the student to insert the plastic spreader and use it to spread the bacterial broth so that is completely covers the surface of the agar.  The lid of the plate is then closed again and the spreader is placed into the Discard Pot of disinfectant.  The agar plate is now a bacterial lawn plate.

• The lawn plate should now be left for 5-10 minutes to allow the broth to be absorbed into the agar surface.

 Placing the Antibiotic Discs:

• Students should now take the forceps and pass the end of them through the Bunsen Burner flame a few times.

• Using the forceps, a filter paper disc is picked up and is dipped into an antimicrobial test solution.  The disc is shaken to remove excess drops of the test solution and the lid of the bacterial lawn plate is lifted slightly.  The filter paper disc is then placed in the correct area of the plate (according to the labelling of the underside of the plate).  The forceps can be used to lightly press the filter paper disc to ensure good contact with the agar surface is made.  The petri dish lid is then closed, and the forceps are passed through the Bunsen Burner flame.

• This process is repeated for each of the different antimicrobial substances provided.

• If using an antibiotic Mast Ring, the procedure only needs to be carried out once.

• Students can now use two small strips of tape to secure the lid of the lawn plate to the base.  The students should use the scissors to cut the tape – they must not bite it!  It should also be reinforced that the bacterial lawn plates need to have an aerobic environment to prevent the development of unwanted/harmful microbial cultures so the petri dish should not be completely sealed.

• The students can now clear away their equipment as directed by the teacher in a manner which ensures the equipment trolley is safe for the technician team to collect and dispose of.

• The laminated sheet the students have been working on can be placed into a tray containing Virkon Disinfectant.

• Ensure all syringes and spreaders are placed into the Discard Pot containing Virkon Disinfectant.

• All student lawn plates should be gathered into a tray together, the teacher should check these have all been sealed up correctly and the technician will monitor these whilst they are developing in the incubator.

• All bacterial broths, and associated equipment should be returned to the equipment trolley.

• Once the equipment is all packed away, students can wipe down the benches, wearing gloves, using Virkon Disinfectant.  Students MUST now thoroughly wash their hands using hot soapy water before leaving the laboratory.

• The lawn plates are then placed into the incubator in order to develop.  The incubator should be set to 25°C – 30°C and the plates should be monitored for 24-72 hours, or until visible zones of inhibition can be seen.

Analysis of Zones of Inhibition:

• Once the plates have been incubated, and visible zones of inhibition are seen on the lawn plate, the plates can be returned to the class for analysis.

• The diameters of the zones of inhibition are measured and noted.  The more effective the antimicrobial substance, the wider the diameter of the zone of inhibition.

• During the analysis session, the incubated bacterial plates should be returned to the class, along with an autoclave bag, so that their bacterial plates can be placed inside once their analysis is complete.

• Students should  be reminded to thoroughly wash their hands using hot soapy water after handling the bacterial plates, before they leave the laboratory.

 Possible Learning Extension:

• Antibiotics of different concentrations could be used here for further investigation into the effect of the concentration of the antimicrobial substance on the growth of bacteria.

• In this case, the antibiotic discs would be removed from their cartridges using the forceps as outlined above.

• Students could also produce their own dilutions of one particular anti-microbial substance to further investigate the effects of concentration.  Students would have the opportunity to further demonstrate their use of laboratory equipment by using serial dilution and the associated equipment.

Part 2: Measuring the effects of an antimicrobial substance on the growth of bacterial populations in a broth culture.

Method:

• Students are provided with the following equipment:

  · Marker Pen

  · A4 Laminated Sheet (which has been sitting in Virkon Disinfectant - as used in Part 1)

  · 1 x McCartney Bottle containing E-Coli Bacterial Broth

  · 5 x sterile McCartney Bottles containing 9mls of sterile nutrient broth

  · 10 x sterile 1ml syringes

  · 5 x sterile spreaders

  · 5 x sterile nutrient agar plates

  · Discard Pot containing Virkon Disinfectant

  · Bunsen Burner

• Prepare the working area by cleaning the bench surface wearing gloves using the Virkon Disinfectant.

• Students set up and light a Bunsen Burner so that the yellow safety flame is in operation. They place the disinfected laminated sheet on the bench in front of the Bunsen Burner – this is now the students sterile working surface.

• The sterile McCartney Bottles containing Nutrient Broth are labelled using the marker pen with the following dilutions:

  · 1/10

  · 1/100

  · 1/1000

  · 1/10000

  · 1/100000

• The Bunsen should be switched to the roaring flame.  Students then remove the lid of the McCartney Bottle containing the E-Coli bacterial broth and flame the neck of the bottle as they did in the previous part. Using a sterile 1ml syringe, 1ml of E-Coli broth is drawn up and the lid of the McCartney Bottle is replaced.  Without putting the bacterial syringe down, the McCartney Bottle labelled 1/10 is picked up, and the lid is removed.  The  neck of the bottle is passed through the Bunsen Flame as before, and the 1ml syringe is inserted.  The bacterial broth is ejected from the syringe into the sterile nutrient broth, the neck of the bottle is passed through the Bunsen flame and the lid is replaced.  The syringe can be placed into the discard pot containing Virkon solutions, ensuring that the disinfectant is drawn up into the syringe.  The McCartney bottle is gently shaken to mix the culture and it now contains a 1 in 10 dilution of the original bacterial broth culture.

• The above step is repeated using a fresh sterile syringe, the 1/10 dilution bottle instead of the original culture and the 1ml aliquot is added to the McCartney bottle labelled 1/100.

• This is then further repeated for the other dilutions required, ensuring the previous dilution is used to take the 1ml sample from to add to the next dilution.

• On the underside of the 5 x agar plates provided using the marker pen, students should label the base of the plate (around the edge) with their initials, class, date, name of the bacterial culture (E-Coli K12) and the dilution.

• Next, working as closely to the Bunsen Burner as is safe and possible, they should add 1ml of each of their diluted E-Coli broths to the relevantly labelled agar plate and spread the bacteria using the L-Shaped Spreader to cover the surface of the plate as they did in the previous part.

• Once the lawn plate have been left to sit for 5-10 minutes, they can be sealed using two small pieces of sticky tape and can be incubated and monitored for 24-72 hours in an incubator set to 25°C – 30°C.

Analysis of Results:

• After incubation, during the analysis session, the incubated bacterial plates should be returned to the class, along with an autoclave bag, so that their bacterial plates can be placed inside once their analysis is complete.

• Students will need to choose a plate that shows a suitable number (20 -100) of non-overlapping colonies, then count the number of whole colonies that can be seen on each of their dilution plates. Each colony has been formed from one initial bacterium.

• Calculate the number of bacteria in each cm³ of the broth dilution used to inoculate the plate.

• Calculate the number of bacteria in each cm³ of the original undiluted broth culture that was incubated with an anti-microbial chemical. This is the population density.

• The population density of the untreated broth culture should also be estimated, to see the effects of the anti-microbial chemical.

• You can extend the activity by allowing each group in the class to carry out serial dilutions of broth cultures that have been treated in different ways with the anti-microbial chemicals.

Calculating the Population Density:

• Once the students have identified the most suitable plate to count, count the colonies using a permanent marker to cross of the colonies as they count them.

• It may be easier to count in sets of 10 or use a tally chart.

• Count the individual colonies, if colonies have begun to merge then count these as 2 separate colonies.

• Once you have the number of colonies the population density can be calculated using the following formula:

  Number of Colonies x Dilution Factor = Population Density of the Sample.