EXPERIMENTS TO SHOW THE GROWTH OF BACTERIA - basic techniques

(similar techniques are used to grow fungi such as moulds and yeasts)

Bacteria will grow on practically any source of organic food which provides carbon compounds to be respired for energy, and nitrogen compounds to be incorporated into proteins for growth. These substances are normally provided dissolved in water. However, in nature, bacteria can break down solid and insoluble substances by releasing enzymes into the substrate in which they are growing. These substances are thus broken down or digested to simpler substances and the process is called extracellular digestion because it takes place outside the bacterial cells.

Broth The two normal media used in bacteriology are a clear soup-like liquid nutrient broth, usually in tubes, and nutrient agar, which is set into a jelly by the addition of a seaweed extract called agar, and when melted poured into glass or plastic Petri dishes - also known as "plates".

Sometimes, substances are mixed into media, in order to suppress growth of other types of bacteria. There are many such selective media.

A standard carbon source is glucose, and nitrogen is often provided by peptones (partially digested proteins), or inorganic salts. Minerals and vitamins may also be provided, according to the growth requirements of the bacteria. Combinations of chemicals (buffers) may be used to keep the pH stable. Measured amounts of the concentrates are added to water, and dissolved to reconstitute the media.

These media must then be sterilised by heating in an autoclave (like a pressure cooker) at 121°C (pressure 1 bar or 15 lb/sq. in.) for 15 minutes, which kills all living organisms, including spores.

All apparatus used from this point onwards must be sterilised by heat (glassware - 160 °C for 2 hrs) or exposure to radiation.

Aseptic techniques must be used to reduce the likelihood of bacterial contamination. This usually involves disinfection of working areas, minimising possible access by bacteria from the air to exposed media, and use of flames to kill bacteria which might enter vessels as they are opened.

Bacteria may be introduced to the media (inoculated) by various means. Usually the bacteria e.g. from a drop in a heat-sterilised loop are spread on the surface of (ready set) agar. A similar technique is used with broth cultures.

Sometimes the bacteria are introduced using a sterile pipette to the Petri dish before the (fairly cool) agar medium is poured on top ("pour plates"). Then the Petri dishes containing agar or tubes containing broth are incubated, i.e. put in a special apparatus at a fixed temperature (usually 37°C - human body temperature, for possible pathogens - or 25°C for bacteria from the environment). In schools, lower incubation temperatures are used in order to discourage the growth of potential pathogens. When growing bacteria, it is usual to invert the Petri dishes, so as to prevent condensation droplets from falling onto the surface of the agar. Petri dishes are often "sealed" at this stage to prevent people who handle them from contamination by bacteria, which will multiply greatly. It is normal to use 2 strips of adhesive tape from base to lid rather than attempt seal the circular edge of the Petri dish. This is to guard against the possibility of anaerobic organisms growing due to lack of air. However, it must be borne in mind that any drips from a partially sealed Petri dish are potential sources of infection.

Sealed

Cultures are usually examined after 24 hrs incubation.

Liquid media such as broth become cloudy if bacteria are present. This could be the result of only one bacterial cell originally entering the medium, then dividing repeatedly to produce millions! Cloudy

Bacteria on agar "plates" become visible as distinct circular colonies; each colony should represent an individual bacterial cell (or group) which has divided repeatedly but, being kept in one place, the resulting cells have accumulated to form a visible patch.

By an extension of this method using serial dilutions in sterilised liquids, the number of bacteria in a given amount of sample, e.g. food, can be calculated.

After use, bacterial cultures, etc. must be sterilised by the use of heat, before disposal.

STREAKING

Purpose

This procedure is used in order to check

1) whether a culture consists of only one organism ( a "pure culture") or if there are more than one organisms in it (contaminated)

2) whether a culture is viable, i.e. able to grow (perhaps a "stock" culture kept for some time in the fridge)

Background

The basic tools used in this procedure include a (sterile) "loop", and a Petri dish containing an appropriate agar, well cooled and set, with a dry surface. The principle is that individual microbial cells can be separated by dragging them over the surface of the agar, and then given a chance to grow into individual colonies

Materials

Petri dish containing (set - fairly firm - dry) nutrient agar.

Wire loops, mounted in metal handle

Inoculum material in broth or other (liquid) medium

Procedure

Clear an area of bench, and wipe it with disinfectant.

Flame a wire loop, bringing it all to red heat, and leave it upright in a rack to cool.

(Optional - repeat to get several cooled loops)

streaking pattern

1) Using the loop, take a drop of the liquid culture medium (broth) provided and spread it carefully in a line across the surface of the agar as shown. With the same loop, a second, third and fourth line may be drawn parallel to the first. Close the lid of the Petri dish immediately.

2) Sterilise the loop in the flame once again, and allow it to cool.

3) Turn the Petri dish so that the end of the previous lines can be the start of the next ones.

4) Take a cooled loop and make 2 or 3 strokes as before. Close the lid of the Petri dish immediately.

Repeat 2,3,4 until there is no more space round the edge (4 or 5 times), then finish off with a single zigzag streak across the middle.

Seal and label the Petri dish with the culture reference and your name and the date. Place it in an inverted position in the incubator at an appropriate temperature.

Please turn over for the completion stage.

Results

After an appropriate time:

Examine the Petri dish WITHOUT OPENING IT and look for individual colonies. Draw it.

space to draw your own plate

Has the technique caused the micro-organisms in the culture to become separated?

Was the original culture viable?

Was the original culture a pure culture?

How would you obtain a pure culture from your plate?

DILUTION PLATING

Purpose

This procedure is used to identify the number of viable micro-organisms in a fixed amount of a liquid. It can also be fairly easily modified to give results with solid substances, e.g. macerated foods.

Background

Serial dilution involves repeatedly mixing known amounts of source culture with (sterilised) liquid. 1 ml added to 9 ml gives a 10-fold dilution; 1 ml added to 99ml gives a 100-fold dilution.

When fixed amounts of this dilution series are mixed with an appropriate agar and incubated, then different numbers of colonies will be obtained.

By working back from an easily counted plate and using the appropriate dilution factor, the number of micro-organisms in the original source culture can be calculated.

Procedure

For this exercise, yeast suspensions, ("fresh" or "stale") milk , or water may be used.

Lay out and label the tubes and (empty) Petri dishes as shown in the diagram below.

Each member of the team can take it in turns to perform the repeated sections below, and prompt others as required.

Flame and loosen the lids of tubes 0 and 1.

Using a sterile pipette HANDLED ASEPTICALLY transfer 1 ml of liquid from tube 0 to plate 0, and USING THE SAME PIPETTE, transfer 1 ml of liquid from the source culture (tube 0) to tube 1.

Then : DISCARD THE PIPETTE.

Flame the edge of tube 1. Seal and mix the contents gently.

Repeat the process with the next tube and plate:

Flame and loosen the lids of tubes 1 and 2.

Transfer 1 ml of liquid from tube 1 to plate -1, and also into tube 2.

DISCARD THIS PIPETTE. Flame the edge of tube 2. Seal and mix the contents gently.

Repeat the same steps, 5 or 6 times, moving along the chain as shown in the flow chart below.

At the end of this process:

Take a bottle of sterilised agar from the 45 °C waterbath, where it has been kept just above setting temperature. Dry the outside of the bottle, and flame the top and neck area.

Then WORK QUICKLY AND ASEPTICALLY:

Opening each Petri dish lid only slightly, pour nutrient agar into the dilution liquid already in the Petri dish, until it covers about two thirds of the area - although this is not critical.

Mix the agar with the dilution liquid by a gentle swirling action, then flame the mouth of the bottle and move on to pour another Petri dish. When the bottle is empty, wash it out with hot water.

Leave the dishes undisturbed FLAT ON THE BENCH to set - at least 10 minutes. Check the labelling.

Seal and invert the Petri dishes, and place them in the incubator at an appropriate temperature.

Results

After an appropriate time:

Examine each Petri dish WITHOUT OPENING IT and look for individual colonies.

Some will have more colonies than you can count. Some may have none.

Several intermediate ones will be countable. COUNT AND RECORD these in a table, together with the relevant dilution factors.

Dilution	10⁰	10^-1	10^-2	10^-3	10^-4	10^-5
No of colonies	.	.	.	.	.	.

Is there a general pattern, or mathematical relationship between them?

This procedure is sometimes said to give the number of "colony-forming units" rather than the simple number of viable organisms. What is the difference?

Calculate the number of viable organisms in the original culture.

Show your working.

Think about the units in which your results are presented.