Name : Norbert Ha How Ong, Norisha bte Othman

Group : MB0702
Date : 26th April 2007
Module name: MicroBiology A
Exp no. : 2 (I)

Title
Aseptic broth transfer and colony selection

Objectives
1. Perform basic bacteriology transfer techniques using broth and agar cultures.
2. Handle bacteriology cultures aseptically

Summary of Theory

Media preparation
Aseptic technique is a technique to prevent contamination of cultures. Culture media are
prepared in either a liquid (broth) or solid form. Meticulous care is taken to provide the
proper concentration of nutrients , pH and other factors needed to cultivate
microorganisms.

Glassware such as test tubes containing media are stoppered with cotton plugs or loose-
fitting autoclavable plastic or metal caps to prevent the entrance of foreign
microorganisms. The tubes with their contents are then sterilized. For Petri dishes , they
are sterilized in the autoclave and media are then poured aseptically into them.

Two general-purpose media are nutrient broth and nutrient agar. The broth preparation
contains beef extract and peptone, dissolved in distilled water. Nutrient agar contains all
of the ingredients found in nutrient broth and the solidifying agent agar.

Growth patterns of bacteria

Broth media:
Bacteria may exhibit a particular form of growth such as clouding of the medium
(turbidity), accumulations of cells at the tube bottom (sediment), and form a thin
surface film (pellicle).

Agar media:
Bacteria form visible accumulations of cells which is known as colonies. The
appearance of colonies as to pigmentation, shape , elevation and the patterns of growth.
Procedures

A. Broth Transfer

B. Agar Slant as Sources of Inoculum

C. Inoculation of an Agar Slant

D. Colony Selection

Hypothesis
For experiment A, B and D, growth will be expected which causes turbidity of the
medium. It will accumulate at the bottom of the tube which will form pellicle.

For experiment C. , growth will be expected as colonies on the agar slant.

Discussions

No. Sources of error Improvements

1. Using both hands to remove the caps Ask help from our partner to remove
from the tubes. the cap while one hold the inoculating
loop.
2. Contamination of inoculating loop. Sterilize the inoculating loop and keep
it close to the flame at all times.
Answers to questions

1. 4 specific characteristics of broth cultures are it is in liquid form, contains beef

extract and dissolves in distilled water. After inoculation of broth media, bacteria
may exhibit a particular form of growth.

2. A bacterial colony is a cluster of organisms growing on the surface or within a

solid medium, usually cultured from a single cell and are genetically identical.

3. A nutrient broth contains beef extract and peptone dissolved in distilled water. In
which, peptone contains a mixture of amino acids, carbohydrates, mineral salts
and polypeptides.
A nutrient agar contains all of the ingredients found in the nutrient broth. It also
contains a solidifying agent, agar, generally in 1.5 to 2.0% concentration, which
makes it different form the nutrient broth.

4. 2 ways to sterilize laboratory media are sterilizing by flaming and sterilizing

objects with ethanol.