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The objective of the serial dilution method is to estimate the concentration (number of colonies, organisms, bacteria, or viruses) of an unknown sample.

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.

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.