GENERAL MICROBIOLOGY

METHODS OF CULTIVATION AND INSULATION OF BACTERIA

OBJECTIVES
Let the student know the different techniques of isolation in solid culture media to
obtain pure cultures of bacteria. That the student prepare cultivation means of
general use, differential and selective for the cultivation of different bacterial
species.
INTRODUCTION
Bacteria should be cultured in laboratory culture media to characterize their growth,
make their identification and determine their metabolic activities. A culture medium
is an aqueous solution of different compounds containing all the indispensable
elements that microorganisms require to grow.

Bacteria are usually inoculated or introduced into liquid media (broths) or solidified
with agar for propagation and / or preservation, as well as to study their growth
characteristics. It is important to remember that inoculation of microorganisms in
culture media should always be carried out in an aseptic zone (near the burner or
in a laminar flow hood), conditions that limit the presence of contaminating
undesirable microorganisms.
Isolation techniques allow microorganisms to be obtained from complex samples
(soil, water, food, etc.) in which there is a high microbial diversity, as well as to
check the purity of the cultures obtained. The pure cultures are formed by a single
type of microorganism; And are indispensable to know the morphological
characteristics, staining properties, biochemical activity, pathogenicity, sensitivity to
antibiotics and identification of microbial species.
Isolation of microbial cultures can be done by dilution methods, in solid media by
slab striation or in liquid media. In the first case it is considered that each bacterial
cell that separates will give rise to a population that will form a characteristic
colony, visible to the naked eye.
The main limitation of these dilution techniques is that it is not practical for isolation
from mixtures where the microorganism of interest is in small quantities, where
only the dominant bacteria will be obtained.
In order to favor the isolation of low density cultures, special culture methods are
used, in which media containing special nutrients, antibiotics, high salt
concentrations and / or conditions of pH, light or temperature are used to promote
the growth of the microorganism Of interest and are known as selective or
enrichment.
Other components can be added to the culture media, such as blood, dyes,
indicators, etc. To distinguish different bacterial species by the way they metabolize
the substrates manifested by changes in the appearance or modification of the pH
of the culture medium. These media are known as differential media and are of
great use for the characterization and identification of bacterial species.

MATERIALS
3 boxes of Petri with nutritive agar (Annex 2.2)
2 boxes of Petri with methylene blue eosin agar EMB
(Annex 2.3)
2 Petri dishes with Staphylococcus Agar 110 (Annex
2.5)
2 Boxes of Petri with Minimum Medium of salts (Annex 2.3)
1 tube with sterile distilled water
2 tubes with Nutrient Broth (See Annex 2.1)
4 tubes with Nutritive Agar (See Annex 2.2)
1 Lighter
1 Inoculating handle
1 stirring grill
1 Autoclave
PROCESS
The professor will provide pure cultures of Bacillussubtilis, Escherichia coli,
Staphylococcus aureus, Pseudomonas fluorescens, Klebslellasp. And
Streptococcus spp.
It will also provide them with a problem sample per team (with two different
microorganisms) in which they will practice the cross-striation isolation technique.
ROSS STRESS TECHNIQUE
A.-In the back and outside of the box, divide it into four quadrants. Take a sample
with the previously flamed and cold handle, inoculate the sample making 4-5 single
grooves closely together from side to side on the first quadrant of the box, close
the box (Figure 4).
B. Flame the inoculating handle and spin the Petri dish a quarter of a turn. Open
the carton again and cool the sewing handle by touching the surface of the medium
away from the area of freshly made grooves.
C.-Touch the handle once the surface of the original set of grooves and make a
second group of grooves in the second quadrant as in the previous case.
D.- Repeat procedures 4 and 5 in the third quadrant and when sowing in the last
quadrant, the sowing loop should not be flashed and a more open (simple) groove
made.
SOWING IN TUBES WITH CALDER AND NUTRITIVE AGAR
Sow a different pure culture in each of the two tubes with nutritive broth and in
tubes with nutritious inclined agar.
Sow with the extended handle the same pure cultures in two other tubes with
nutritive agar solidified in straight form (Figure 5).
SOWING OF PETRI BOXES WITH DIFFERENTIAL AND SELECTIVE MEANS

1.- Divide in three parts, a Petri dish with EMBAgar, another with Agar 110yuna of
MM. Sow in each part a different pure culture by simple stria.
2.- In another series of three boxes with each of the means described above,
cross-streak the problem sample.
INCUBATION CONDITIONS
1.- Incubate the boxes inverted at 35 C for 24 hours. From the boxes inoculated
by crossed striae, select the most isolated colony (usually from the fourth
quadrant). Transfer a small sample of this colony to lean tubes with nutritive agar.

2.-Incubate the tubes at 35 C for 24-48 hours. Observe the appearance of

colonies and report how they are distributed a long the tube.