Separating micro-organisms

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Antibody (Antiserum) is dropped in the central well and antigen (serum) from a variety of animals is placed in surrounding wells.
If the antibodies meet recognizable antigens, the proteins react and precipitate, giving a visible line.
Whether a reaction occurs depends on the evolutionary relatedness of the animals, so that sheep antiserum reacts with goat serum but not with mouse serum. This point illustrates the specificity of the antibody/antigen reaction.

Set up TWO plates as shown here: note which antibody you put in the central well

Note which Antigens you put in wells 1  8:
Antigen 1 =
Antigen 2 =
Antigen 3 =
Antigen 4 =

Antigen 5 =

Antigen 6 =
Antigen 7 =
Antigen 8 =

* Remember to include a control

Source: Based on material from biology4all website

Determine the effect of chemical agents on bacterial growth.

  1. Describe the physical effect on the growth of bacteria on solid nutrient agar.

  2. Evaluate the effectiveness of commonly available antiseptics and disinfectants.

  3. Discuss the inaccuracies that are inherent in the filter paper disk method.

You will study how certain commonly available disinfectants and antiseptics affect the growth of two common bacterial species.
Disinfectants are described as antimicrobial agents used on inanimate objects such as instruments (medical or dental), plastics or surfaces such as kitchen worktops, toilets, washbasins etc). Disinfectants should remove pathogenic organisms.

Antiseptics are antimicrobial agents that are used on living tissue such as skin.
Disinfectants and antiseptics do not always sterilise because these compounds usually do not kill all fungal and bacterial spores and vegetative bacteria and fungi. Organic compounds e.g. dirt, grease … etc. can interfere with their action reducing the efficiency of the antimicrobial agent.

This practical enables you to study some commonly available antiseptics and disinfectants and assess their efficiency against two common bacteria that can be isolated from kitchens, hospitals and from humans. You can use household products such as Dettol or you can vary the experiment to compare antibacterial soaps, skincare products such as facewashes etc, toothpastes, mouthwashes…….

Useful references on the internet
Lister and antiseptics

E. coli


four disinfectants or antiseptics (can bring from home)

overnight broth cultures of:

Escherichia coli

Staphylococcus epidermis albus

sterile nutrient agar plates (x2)

sterile filter paper disks


glass spreader

  1. Using a sterile pipette remove 0.1 ml from the broth culture of Escherichia coli, and inoculate one labelled nutrient agar plate. Spread evenly to obtain confluent growth after incubation.

  2. Do the same with the other agar plate, using the broth culture of Staphylococcus epidermis albus.

  3. Label the four antiseptics or disinfectants that you will be testing A, B, C and D.

  4. On the bottom of the agar plates, mark four sectors using a marker pen. Label the sectors A, B, C and D.

    • These sectors correspond to the letters you put on the antiseptic or disinfectant containers.

  1. Sterilise the tip of your forceps by passing it through the flame of the Bunsen burner two or three times.

  2. Aseptically pick up a sterile filter paper disk with your sterile forceps and dip the disk into the disinfectant or antiseptic labelled A.

    • Be sure that the excess disinfectant has drained off.

  1. Place the disk in centre of Sector A of the Staphylococcus epidermis albus inoculated plate.

  2. Using the same disinfectant, place another disk in Sector A of the E. coli-inoculated plate.

    • You are comparing the effectiveness of each disinfectant or antiseptic on both organisms.

  1. Repeat steps 8 and 9, placing the other disinfectants in the other sectors.

  2. Gently press the disks down with the tip of your flamed forceps to ensure contact with the nutrient agar.

  3. When all four disinfectant-soaked disks have been placed in all four sectors of both plates, diametrically seal them with tabs of tape, invert the plates and incubate them at 30C for 48 hours.

12 Observe, measure, and compare the zone of no growth (inhibition), if any,

around the disk for each disinfectant or antiseptic for both organisms.

13 Evaluate the effectiveness of each of the antiseptics/disinfectants at

controlling the growth of the microbes used here.

14 Describe the usefulness of this experiment in terms of controls, reliability, &

possible inaccuracies.

Source: Bio-Rad ‘pGLO Bacterial Transformation Quick Guide’
Available online at

Transformation Procedure

The aim of this experiment is to transform E.coli bacteria to express the green fluorescent protein. The pGLO plasmid is used as a vector in this experiment.

  1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with your group’s name. Place them in the foam tube rack.

  1. Open the tubes and, using a sterile transfer pipette, transfer

250 l of transformation solution (CaCl2) into each tube.

3 Place the tubes on ice.

4 Use a sterile loop to pick up a single colony of bacteria from your starter

plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the –pGLO tube.

5 Examine the pGLO DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on ice. Also close the –pGLO tube. Do not add plasmid DNA to the –pGLO tube. Why not?

6 Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.

7 While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom (not the lid) as follows:

    • Label one LB/amp plate: +pGLO

    • Label the LB/amp/ara plate: +pGLO

    • Label the other LB/amp plate: -pGLO

    • Label the LB plate: -pGLO

      1. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and (-) pGLO tubes into the water bath, set at 42C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water.

When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0C) to 42C and then back to the ice must be rapid.

Incubate tubes on ice for 2 minutes.

9 Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipette, add 250 l of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipette for the other tube. Incubate the tubes for 10 minutes at room temperature.

10 Tap the closed tubes with your finger to mix. Using a new sterile pipette for each tube, pipette 100 l of the transformation and control suspensions onto the appropriate nutrient agar plates.

Transformation plates

Control plates

11 Use a new sterile loop for each plate. Spread the suspensions evenly

around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR.


LB/amp LB/amp/ara LB/amp LB

12 Stack up your plates and tape them together. Put your group name on the bottom of the stack and place the stack of plates upside down in the 37C incubator until the next day.

Source: Adapted from NCBE Practical Biotechnology

A similar protocol using lettuce leaves is available at

Protoplasts are plant, fungal or bacterial cells which have had their cell walls removed; usually by enzymic digestion. The resultant ‘naked’ cells can be used in techniques such as the creation of transgenic plants.


1 lettuce leaf (from a round lettuce)

0.1 cm3 21% sorbitol solution

20cm3 13% sorbitol solution

0.5cm3 Viscozyme (mixture of carbohydrase enzymes)

Paper tissue

10ml syringe

2 x 1ml syringe

Test tube

Centrifuge tube

Stirring rod

Filter funnel

Nylon gauze to plug filter funnel

Microscope slide

Cover slip

Microscope with x40 objective

Water bath set at 37oC

Bench centrifuge

Preparing the lettuce
1. Cut the lettuce leaf into pieces roughly 5mm x 5mm

2. Add 15-20 lettuce pieces to 9.5cm3 13% sorbitol solution in a test tube

3. Incubate the tube at 37oC for 5 minutes.

Digesting the cell walls
4. Gently stir 0.5cm3 of viscozyme into the sorbitol and lettuce preparation

5. Return the tube to the water bath for another 20 minutes. Gently stir the contents from time to time.

Recovery of the protoplasts
6. Tightly pack the spout of the filter funnel with nylon gauze

7. Pour the digested lettuce suspension into the filter funnel

8. Wash any trapped protoplasts through the filter using 10cm3 of 13% sorbitol solution. Collect all the filtrates in a centrifuge tube.

9. Centrifuge the filtrate for approximately 5 minutes at 2000 rpm

10. Carefully pour off the supernatant, leaving a pellet of protoplasts at the

bottom of the tube.

11. Resuspend the pellet in approximately 0.1cm3 of 21% sorbitol solution.

Examining the protoplasts
12. Put some resuspended protoplasts on a slide and gently lower a coverslip into position. Protoplasts can easily be seen without staining using a x40 objective lense.

Source: James Watt College
This technique is used to estimate the number of phage virus particles in a stock suspension. Plaques are clear areas in a lawn of bacteria caused by the lysis of a phage-infected bacterial cell.
24 hour broth culture of E.coli B

Suspension of phage virus (eg Philip Harris Bacteriophage T4B)

8 sterile nutrient broths (4.5 ml each)

Gilson pipette

Sterile blue tips

10 tubes sterile sloppy (0.7%) nutrient agar: 6mls each

(NOTE: these must be kept at 45oC).

10 nutrient agar plates

Water bath at 45oC

Serial diluting the phage
1. Use sterile nutrient broth to prepare serial dilutions of the stock phage

ranging from 10-1 to 10-8 (use 0.5ml phage and 4.5ml broth each time).

Sterile nutrient broth (4.5ml per tube)

Combining the bacteria and virus
NOTE: The following steps use sloppy agar. This will start to set if the

temperature is allowed to drop below 450C. These tubes must be

kept warm at all times.
2. Label each of the tubes of sloppy agar with a concentration ranging from 10-1 to 10-8.

Also label a tube as ‘stock’ and another tube as ‘control’.

3. Add 0.4ml E.coli B to every tube.
4. To each tube add 0.2ml appropriate phage dilution: remember to add undiluted phage to the tube labelled ‘stock’ and to add nutrient broth to the tube labelled ‘control’.
5. Rapidly mix by rotating the tubes between the palms of your hands.

Pouring the plates
6. Label the nutrient agar plates from 10-1 to 10-8, plus ‘stock’ and ‘control’ plates.
7. Pour the contents of the appropriate tube (see above) over the surface of a nutrient agar plate so it forms an even layer.
8. Allow to set and then invert & incubate for 28-24 hours at 30oC.

Estimating the number of phage
Some plates will have so many plaques they will overlap each other, whilst other plates (at higher dilutions) will have very few plaques. At least one plate will have plaques in countable numbers.
9. Find the plate or plates with plaques in countable numbers.
10.Count the number of plaques & multiply this figure by the dilution factor to obtain the concentration of the phage.

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