In view of the importance of microbial activities in practically all aspects of everyday life it is highly desirable that students should study microbiology in a practical way. There are plenty of very worthwhile and rewarding practical microbiology investigations which are relatively reliable, easy to carry out, inexpensive, and suitable for quantitative work at GCSE level.
Provided that simple guidelines are followed, which involve no more than normal good laboratory practice and the observance of aseptic techniques, then teachers need not be deterred from embarking on microbiology because of the perceived safety concerns.
There are, of course, risks associated with practical microbiology because of the impossibility of being certain that pathogens are not inadvertently cultured. Therefore all cultures must be treated as if they are pathogenic, and must be handled and disposed of safely. To discourage growth of pathogens, cultures should not incubated at temperatures above 30 °C.
It is essential that teachers adhere to current and safety regulations such as COSHH and that they consult the following publications, details of which are given under 'Literature'.
Safety in Science Education HMSO 1996
Topics in Safety Association for Science Education 1988
Any guidelines issued by School Governing Bodies and LEAs should also be consulted.
A risk assessment should be carried out in respect of each item of practical work and any appropriate action be taken.
Aseptic techniques, fully described in most relevant standard texts, should invariably be adopted. These cover procedures before, during and after the practical sessions and are designed to prevent accidental contamination of cultures and the escape of microorganisms from them. Particular care must be taken with the disposal of live cultures.
The above requirements may well appear to be formidable but, once required procedures have been gone through, good working practices established and vigilance maintained, practical microbiology presents no more difficulty for the teacher than do most other areas of practical biology.
There are numerous sources of advice, reassurance and help, such as MISAC and CLEAPSS (details under 'Useful Addresses'). Particular reference should be made to the CLEAPSS Handbook, sections 15.2 and 15.12.
Five investigations are described; they have been found to work well under class conditions and demand few resources. Most provide ample scope for manipulating variables and could be formatted into an Sc1 framework. The risks associated with these five investigations are extremely low.
List of practical activities
1. Testing milk for freshness.
2. A look at personal hygiene.
3. Testing the effect of antibiotics on bacteria.
4. Culturing bacteria from different sources.
5. Testing solid surfaces for microorganisms
Technicians' information
Assuming 15 working groups
It is assumed that students will collect their samples of milk and the methylene blue solution from one of five 'stations' around the laboratory, minimising the amount of glassware needed.
At each 'station'
100 cm3
of each of the following milk samples, freshly poured into clean beakers or other containers, each provided with a sterile plastic syringe for dispensing 10 cm3
.
Each container labelled by letter:
A pasteurised, freshly purchased, previously unopened
B pasteurised, previously unopened, but stored for 2 days at room temperature
C pasteurised, opened to the air and stored at room temperature for 2 days
D as C, but boiled for 3 minutes and then cooled, just before the practical
E UHT, previously unopened
50 cm3
of methylene blue solution in a sterile beaker or other container, the container provided with a sterile plastic syringe for dispensing 1 cm3
, and labelled 'methylene blue solution'. Dissolve 1 methylene blue tablet (available from BDH Ltd: see 'Useful Addresses') in 200 cm3
of distilled water in a sterile container and make up to 800 cm3
with more distilled water.
Per working group
5 clean test tubes, with rubber bungs to fit
test tube rack, suitable for immersion in water bath
marker, for writing directly on to tubes
Per class
thermostatically controlled water bath(s), sufficient to accommodate all test tube racks: set at 30°C
sight of clock; or stopclock per working group
Note
The redox indicator, resazurin, may be used instead of methylene blue.
Dissolve 1 tablet in each 50 cm3
of distilled water on the day of the practical. It is blue in fresh milk but as the pH reduces, it becomes pink. Finally it changes to white as the oxygen content is lowered by bacterial activity. There is thus a three-colour scale for assessing freshness.
Milk is an ideal food source for certain bacteria and, depending upon how fresh it is and how well it has been stored and processed, milk may contain large numbers of these bacteria. You are provided with 5 different samples of milk, to be tested for their bacterial content.
The method you will use is very similar to that used by dairies and public health inspectors.
When methylene blue is added to milk containing large numbers of bacteria, the dye gradually becomes decolourised. This is because the dye is chemically changed by a substance produced by bacteria as they respire.
You will add a certain volume of blue dye to equal-sized samples of milk and see if the dye is decolourised and, if so, how long it takes. The mixtures of dye and milk will be kept at a temperature of 30 °C, to speed up the respiration rate of any bacteria.
Apparatus and materials
1. The following milk samples, to be collected from one of the 'stations' as directed by your teacher:
A pasteurised, freshly purchased, previously unopened
B pasteurised, previously unopened, but stored for 2 days at room temperature
C pasteurised, opened to the air and stored at room temperature for 2 days
D as C , but boiled for 3 minutes and then cooled, just before the practical
E UHT, previously unopened
2. methylene blue solution
3. five clean test tubes with rubber bungs
4. test tube rack
5. marker, for writing directly on to tubes
6. thermostatically controlled water bath(s), set at 30 °C
7. sight of clock, or stopclock
A Label the test tubes A to E,
B Use the syringe to measure l0 cm3
of each milk sample into its correct tube.
C Use the syringe to measure 1 cm3
of methylene blue dye into each tube of milk.
D Close each tube with a stopper, then gently turn the tubes upside down two or three times to mix the contents.
E Place the tubes together in the water bath, then note and record the time, or start a stopclock.
F Design and draw up a table in which you will record your observations of colour for each tube, at l0 minute intervals, for up to an hour.
G Observe the tubes every ten minutes, but do not shake them. The samples are said to be decolourised when they are white to within 5 mm of the surface.
H Record how far the contents of each tube have become decolourised (e.g. 'completely blue', 'faintly blue', 'bottom 2 cm white, rest blue'). Continue for at least 40 mins, but up to an hour if possible.
I Explain your observations for each milk sample.
This investigation uses the harmless yeast Saccharomyces cerevisiae to simulate the contamination of hands with faecal bacteria and the effectiveness of hand washing in removing them. Different brands and thicknesses of toilet paper, and different types of soap, including antiseptic soaps, may be evaluated.
Technicians' Information Assuming 15 working groups
Per working group
First lesson
3 malt extract agar 'lawn' cultures of Saccharomyces cerevisiae. The materials are available from biological suppliers. The required preparation, using aseptic technique, consists of pouring malt extract agar plates, and inoculating the solidified agar with a pure culture of Saccharomyces cerevisiae, distributing it evenly using a glass rod 'spreader' then incubating for 2-3 days at 25 °C.
3 previously-poured malt extract agar plates, stored upside down so that the surface remains dry
different brands of toilet paper
different brands or types of soap
autoclavable plastic bag for disposal of used cultures and toilet paper
Bunsen burner to create updraught (as part of the aseptic technique which must be used for this practical)
marker pens
tape to secure lids to plates
arrangements for all students to wash their hands thoroughly using hot water and soap, and then to dry them using clean paper towels
Per class
incubator, set at 25 °C
Second lesson
suitable container for disposal of used plates
Safety Note
Even though all plates will be destroyed unopened, it is important that students wash their hands thoroughly as directed, in order to reduce the risk of inadvertently culturing organisms already present on the skin surface. An alternative or additional measure would be the use of sterile gloves.
All plates should be sealed completely, after incubation, before the second lesson, with tape around the circumference, in case skin bacteria have been incubated inadvertently.
This investigation uses a harmless yeast Saccharomyces, to mimic the bacteria present in faeces, so as to show possible contamination of hands after using the toilet. It also examines the effect of hand washing in removing microbes.
Different brands and thicknesses (i.e. using one or two sheets) of toilet paper, and different types of soap, including antiseptic soaps, may be tested. Your teacher will suggest which to use.
You will be provided with three agar plates containing harmless yeast Cells. You will then see whether toilet paper and hand washing prevent these yeast cells from being transferred to a sterile agar plate by your hands.
The diagram shows a summary of the procedure. The diagram includes 'blank' Petri dishes in which you may wish to make drawings of your results.
Apparatus and materials
1. 3 cultures, on agar plates, of Saccharomyces
2. 3 sterile agar plates
3. different brands of toilet paper
4. different brands of soap
5. plastic bag for disposal of used cultures and toilet paper
6. Bunsen burner
7. marker pen
8. tape to secure lids to plates
9. arrangements for washing your hands, using hot water and soap and drying them using a clean paper towel
l0. incubator, set at 25 °C
Method
A Collect together items 1- 8
B. Label the three sterile plates X, Y and Z.
C. Wash your hands thoroughly using hot water and soap, then dry them on a clean paper towel.
D. Open one plate of Saccharomyces and wipe two fingers lightly over the surface.Then open the sterile plate X, touch the agar surface lightly with the same two fingers and quickly replace the lid of X.
E. Wash your hands as before, then wrap two fingers in one or two thicknesses of toilet paper (as directed by your teacher). Open another plate of Saccharomyces and wipe the two fingers lightly over the surface. Try to wipe your fingers across the culture in as similar way as possible to step D. Remove the toilet paper and place it in the bag provided. Then open the sterile plate Y, touch the agar
surface lightly with the same two fingers and quickly replace the lid of Y.
F. Repeat step E but, after removing the toilet paper, again wash your hands thoroughly using the type of soap suggested by your teacher, and dry them on a clean paper towel. This time, use sterile plate Z.
G. Use tape to secure the lids on all the plates, invert and label them with your initials, and place them in the incubator, where they will be left, at a temperature of 25 °C, for 3 days.
H Wash your hands thoroughly before leaving the laboratory.
Apparatus and materials
1. Your agar plates, X, Y and Z.
2. A suitable container for disposal of the used plates.
Method
A. Collect your plates X, Y and Z.
B. Count or estimate the number of colonies of Saccharomyces that have grown.
C. Design and draw up a table in which you will record your observations and also to include those from the rest of the class.
Your table may for example, look something like this:
| Number of colonies of Saccharomyces | ||||
| Brand of toilet paper | Soap- brand 1 | Soap- brand 2 | ||
| ............................... | ............... | ............... | ............... | ............... |
| ............................... | ............... | ............... | ............... | ............... |
D. If appropriate, make drawings of your results in the 'blank' Petri dishes shown in the diagram.
E. Give ONE scientific reason and ONE safety reason for washing your hands thoroughly in hot water and liquid in step C.
F. What do the class results suggest about personal hygiene procedures?
Technicians' information Assuming 15 working groups
This investigation uses cultures of two bacteria, Escherichia coli and Staphylococcus albus and examines the effect on them of the antibiotics penicillin (used at two concentrations) and streptomycin, applied to previously prepared seeded plates of the two organisms in the form of impregnated paper discs.
The discs should be handled only with forceps, to avoid the possibility of allergic response. This warning should be repeated to the students.
The materials (cultures of the two bacteria, nutrient broth, nutrient agar, antibiotic discs and blank control discs) are readily available from biological suppliers. The required preparation, for which aseptic techniques must be used, consists of inoculating nutrient broth with the bacteria, and then incubating it for 24 hours. Then seeded agar plates are made by mixing some of the broth culture with liquid nutrient agar, pouring the mixture and leaving the plates to solidify.
The detailed instructions are:
1. Use a sterile loop to inoculate a 15 cm3 vial of nutrient broth with a small quantity of material scraped from the surface of a culture of Escherichia coli. Label the vial and then repeat the procedure, using Staphylococcus albus.
2. Incubate both vials at 30 °C for 24 hours.
3. Prepare seeded plates by mixing some broth culture (3 or 4 drops per plate) with liquefied nutrient agar. Pour the number of plates required for the class and leave them to solidify for a few hours.
4. Seed half of the plates with Escherichia coli Label these plates and then seed the other half with Staphylococcus albus. Label these plates.
After setting up the investigation in the first practical session, the plates should be incubated for 24 hours and then examined as soon as possible. Further activity can be slowed, if necessary, by refrigerating the plates.
The practical may be varied or extended by using discs soaked in materials such as deodorant, disinfectant, skin preparations, etc.
Note
If the time of the second lesson dictates, unwanted progression can be prevented by killing the bacteria. To do this, invert the dish and place filter paper in the lid, moistening it with 40% methanal (formaldehyde) solution. Close the lid and tape it. Leave the dish overnight, then remove the paper and tape up the dish again.
Per working group
1 nutrient agar culture of E. coli, and 1 nutrient agar culture of S. albus, prepared as above.
pair of forceps
1 disc of each of the following, in sterile container:
blank control (without antibiotic)
penicillin 1.5 iu
penicillin 5 iu
streptomycin 25µg
Bunsen burner for flaming the forceps and to create updraught (as part of the aseptic techniques which must be used for this practical)
marker pens
tape to secure lids to plates
safety goggles
Per class
incubator, set at 30°C
Second lesson
graph paper
autoclavable plastic bag for disposal of used plates
You will be investigating the effect of two different antibiotics on two different bacteria. The antibiotics to be tested are penicillin and streptomycin. Both antibiotics are widely used by doctors. The penicillin will be tested in two different concentrations. The bacteria to be used are Escherichia coli (E. colt) and
Staphylococcus albus (S. albus).
First lesson
Apparatus and materials
1. 1 nutrient agar culture of each of the bacteria Escherichia coli (E. coli) and Staphylococcus albus (S. albus).
2. pair of forceps
3. 1 disc of each of the following:
blank control (without antibiotic)
penicillin 1.5 iu
penicillin 5 iu
streptomycin 25 µg
The discs should be handled only with forceps, to avoid the possibility of allergic response.
4. Bunsen burner for flaming the forceps and to create updraught (as part of the aseptic technique).
5. marker pens.
6. tape for securing lids to plates
7. safety goggles.
8. incubator, set at 30 °C.
Method
A. Collect together items 1-7. Put on your safety goggles.
B. Light a Bunsen burner to create an updraught which will help to carry any airborne microorganisms away
from your opened Petri dish containing agar. Position the apparatus you are going to use around the base of the Bunsen burner. Be careful to open the agar Petri dish for the minimum time and do not breathe on it!
C. Sterilise the end of the forceps by holding them in the Bunsen flame briefly.
D. Use the sterilised forceps to transfer, gently, one of each type of disc provided on to the agar surface.
The discs should be positioned as shown in the diagram, to avoid the places where tape will be placed.
Three discs contain an antibiotic; the other is a blank control. Note the position of each type of disc.
Close the dish.
Second lesson
Apparatus and materials
1. Your plates from the first lesson.
2. Sheet of graph paper.
3. Autociavable plastic bag or other container, for your used agar plates.
Method
A. Collect your plates and a sheet of graph paper.
B. Examine the agar surface, looking for clear areas around the discs. Place the plate on the sheet of graph
paper and record the diameter of any clear areas.
C. Place your used agar plate in the container provided, for disposal.
D. Design and draw up a table in which to record your results, and also those from the rest of the class.
You may wish to determine the mean results for the class. Summarise your conclusions.
This investigation examines samples of pond water, tap water, milk and soil for the presence of bacteria.
Unlike the previous investigations, it provides experience in the use of an inoculation loop as part of
aseptic technique.
Technicians' information Assuming 15 working groups
Preparation consists of preparing sufficient nutrient agar plates and providing the required sarnples.
First lesson
Per working group
Access to labelled samples of pond water, tap water, milk and soil 'Mud', collected/prepared in sterile vessels. The 'mud' is prepared by adding a little sterile water to a soil sample.
5 plates containing solidified sterile nutrient agar
wire inoculation loop
Bunsen burner to create updraught (as part of the aseptic technique)
marker pens
tape to secure lids to plates
Per class
incubator, set at 25 °C
Second lesson
suitable container for disposal of used plates
You will be investigating samples of pond water, tap water, milk and soil 'mud' for the presence of bacteria.
First lesson
Apparatus and materials
1. access to samples of pond water, tap water, milk and soil 'mud', collected or prepared in separate containers
2. 5 plates containing solidified sterile nutrient agar (one will be left 'blank', as a control)
3.wire inoculation loop
4. Bunsen burner to create updraught (as part of the aseptic technique)
5. marker pens
6. tape to secure lids to plates
7. safety goggles
8. access to incubator, set at 25 °C
Method
A. Collect together items 1-7. Put on your safety goggles.
B. Light a Bunsen burner to create an updraught which will help to carry any airborne microorganisms away from your opened Petri dish containing agar. Position the apparatus you are going to use around the base of the Bunsen burner. Be careful to open each agar Petri dish for the minimum time and do not breathe on it!
C. Sterilise the wire loop by heating it briefly to redness in the Bunsen flame.
D. Without putting the sterilised loop down, take the stopper out of one of the sample containers. (Use, the hand which is holding the loop to grasp the stopper and the other hand to hold the container). While you are doing this, the loop will cool sufficiently.
E. Collect a drop of the sample in the loop and replace the stopper.
F Lift the lid from an agar plate slightly and at an angle so that the agar is not fully exposed to the air. Quickly spread the drop from your loop on to the agar surface and replace the Petri dish lid. Be careful not to let the wire loop dig into the soft agar.
G. Carefully re-sterilise the loop in the Bunsen flame by heating the loop at its base, near the holder, and slowly drawing it through the flame.
H. Tape the lid to the base, invert, then label the base of the dish with the sample type, the date and your initials
I. Repeat steps C to H, three times, using a different sample each time. Leave the fifth dish unopened, but tape, invert and label it.
J. Place the dishes in the incubator, set at 25 °C.
Second lesson
Apparatus and materials
1. nutrient agar plates from the first lesson
2. container for your used agar plates
Method
A. Collect your plates and examine the agar surface, looking for colonies of bacteria.
B. Make a drawing of the contents of each plate, to show the bacterial colonies on each agar surface. Add
notes, if required, to help describe what the colonies look like.
C. Place your used agar plates in the container provided, for disposal.
D. Summarise your conclusions.
This practical allows students to find out if solid surfaces are contaminated by microorganisms. Allow two
lessons, the first of about 1 hour, the second may be shorter.
Technicians' information Assuming 15 working groups
Preparation consists of preparing sufficient nutrient agar plates and sterile swabs.
First lesson
Per working group
cotton wool swabs
Swabs can be made by wrapping cotton wool round the ends of wooden cocktail sticks. Moist, sterile swabs can then be prepared by placing them in a universal bottle with a little water and autoclaving.
1 nutrient agar plate
1 universal bottle containing three moist, sterile cotton wool swabs
Bunsen burner to create updraught (as part of the aseptic technique)
marker pens
tape to secure lids to plates
plastic bag for saving the swabs
Per class
large plastic bag for disposal of used swabs
incubator, set at 25 °C
Second lesson
container for disposal of used plates
Safety Note
It would be prudent to seal the agar plates totally around the circumference after incubation but before they are returned to students. Unknown organisms will be present.
You will be finding out whether microorganisms can be found on a variety of solid surfaces and, if this is the case, you will also get an indication of the number of microorganisms present.
First lesson
Apparatus and materials
1. 1 nutrient agar plate
2. 1 universal bottle containing three moist, sterile cotton wool swabs
3. plastic bag
4. Bunsen burner to create updraught (as part of the aseptic technique)
5. marker pens
6. tape to secure lids to plates
Method
A. Collect together items 1 - 6.
B. Decide which solid surfaces you are going to take samples from. This may mean that you have to leave the laboratory, for example if you choose a dining table. You must consult your teacher to ensure that the surfaces you have chosen are safe.
Examples of suitable surfaces are:
bench top
door handle
window pane
window ledge
C. Rub a swab over each surface you have chosen. Each swab must be marked and returned to the plastic bag. Try to prevent swabs from touching each other.
D. Light a Bunsen burner to create an updraught which will help to carry any airborne microorganisms away from your opened Petri dish containing nutrient agar. Position the apparatus you are going to use around the base of the Bunsen burner. Be careful to open the Petri dish for the minimum time and do not breathe on it!
E. Mark the base of the Petri dish into four quarters. One quarter must not be used.
F. Take a swab from the plastic bag and lift the lid of the Petri dish slightly and at an angle so that the agar is not exposed to the air. Gently rub the swab onto one of the quarters of the agar surface.
G. Put the used swab into the plastic bag provided for disposal.
H. Repeat steps F and G for each of your samples.
I. Tape the lid to the base of the Petri dish. Invert the dish and label the base of the dish with sample types, the date and your initials.
J. Place the Petri dish in the incubator set at 25 °C.
Second lesson
Apparatus and materials
1. Nutrient agar plate from the first lesson, which will now be sealed completely around the circumference.
2. Container for the disposal of used plates
Method
A. Collect your plate and examine the agar surface, looking for colonies of bacteria or fungal growth.
B. Make a drawing of the contents of your Petri dish, to show the bacterial colonies and any fungal growth. Add notes, if required, to help describe the appearance and the quantity of rnicroorganisrns. Summarise your conclusions.
C. Place your used agar plate in the container provided
