Dilution plating

Introduction
Dilution plating is a simple technique used to estimate the number of heterotrophic
bacteria in an environmental sample. Dilution plating is based on serial dilution of the
sample. Serial dilution involves repeatedly mixing known amounts of source sample or
culture with sterile liquid. In such a scheme, 1 ml of sample added to 9 ml yields a 10-
fold dilution. When fixed volumes of this dilution series are spread onto a solid growth
medium and incubated, different numbers of colonies will be obtained.

Objective:
 to identify the number of colonies in a fixed amount of liquid

Materials:
 Slant (Pure culture)
 Test tubes with 9 ml Nutrient Broth
 Alcohol lamp
 Dry powder Agar and nutrient broth
 Beaker
 Analytical balance
 Deionized water
 Stirring rod
 Magnetic stirrer
 Erlenmeyer flask
 Petri dish
 Autoclavable cellophane
 Autoclave
 Paper
 Cotton plugs
 Rubber bands
 Pipette tips
 Pipettor
 Sterile glass spreader
 mechanical shaker

Procedure:

a. Culture media preparation (@lab notebook)

b. Diluting the samples (Draw)
- Loop is heated until red hot and cooled in air briefly
- slant with pure culture is uncapped
- Tip of tube is run through the flame
- from the slant sample is removed using the sterile loop
-dip the loop to a sterile medium to transfer the sample
 - The tube is reflamed.
- The tube is recapped
- Loop is reheated
-Shake the tube on a mechanical shaker
UNKNOWN INOCULUM
- aseptically transfer 1 ml of sample using a pipettor with pipette tip
-discard the pipette
- Flame the edge of the tube
- capped and mix the contents gently.
-label each tube
-Transfer 1 ml of the suspension from the 10-1 dilution tube to the
second dilution tube (10-2 )
diluted to 10-5

c. Plating the samples

- Transfer 1 ml of the 10-3 and 10-5 dilution to be plated to the culture
media (plated in triplicate)
-spread the sample into the surface
-label each plate
- Incubate the plates for 24 to 72 hours.
Surface colonies

Results and discussion:

In this experiment, spread-plate method is applied. After an appropriate time,

colonies are already visible and increasing in number. Some colonies fuse, leading to
erroneous measurements. Plates from 10-3 dilution are too numerous to count and
plates from 10-5 dilution also has a large number of colonies such as at 24 hours
there are 39 colonies, 48 hours has 492 colonies and 72 hours has 554 colonies.

Conclusion
Some plates have more colonies than you can count and some may be less this is
because of the dilution process. Serial dilutions are needed to reach a suitable
dilution for plating to yield countable colonies and colonies can be easily counted if
the sample were spread evenly and properly on the surface of the culture media.
Moreover, organism in culture is dependent on the growth medium and the
incubation conditions.