2018

Gebze Technical
University
Microbiology Laboratory – Report 5
Turkey

Ebru AKHARMAN
20.04.2018
 The Culturing Microorganisms

AIM

In this experiment, 4 types of media are learned and inoculations are performed on these medyums.
Numerous special-purpose media are available for functions including the following:
1. Isolation of bacterial types from a mixed population of organisms.
2. Differentiation among closely related groups of bacteria on the basis of macroscopic appearance of the
colonies and biochemical reactions with in the medium.
3. Enumeration of bacteria in sanitary microbiology, such as in water and sewage, and also in food and dairy
products.
4. Assay of naturally occurring substances such as antibiotics, vitamins, and products of industrial
fermentation.
5. Characterization and identification of bacteria by their abilities to produce chemical changes in different
media.
In addition to nutrients necessary for the growth of all bacteria, special-purpose media contain both nutrients
and chemical compounds important for specific metabolic pathways in different types of bacteria.

Classification of culture media used in microbiology laboratory on the basis of consistency:

1. Solid medium
solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert solidifying agent.
Solid medium has physical structure and allows bacteria to grow in physically informative or useful ways
(e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria or for determining the colony
characteristics of the isolate.
2. Semisolid media
They are prepared with agar at concentrations of 0.5% or less. They have soft custard like consistency and
are useful for the cultivation of microaerophilic bacteria or for determination of bacterial motility.
3. Liquid (Broth) medium
These media contains specific amounts of nutrients but don’t have trace of gelling agents such as gelatin or
agar. Broth medium serves various purposes such as propagation of large number of organisms,
fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VR broth.
1. General purpose media/ Basic media:

Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient
broth and nutrient agar are considered as basal medium. These media are generally used for the primary
isolation of microorganisms.

2. Enriched medium (Added growth factors):

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched
media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate
agar, Loeffler’s serum slope etc. are few of the enriched media. Blood agar is prepared by adding 5-10% (by
volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar.
 3. Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria
and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment
media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made
selective by addition of certain inhibitory agents that don’t affect the pathogen of interest. Various
approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a
combination of these.

4. Differential/ indicator medium: differential appearance:

Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony
colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that
utilize them appear as differently coloured colonies.

Such media are called differential media or indicator media. Differential media allow the growth of more than
one microorganism of interest but with morphologically distinguishable colonies. Then, inoculations are
performed on medyum that have above properties. Inoculating mediums can be listed as follows.

Selective Differential Enriched

Medium
Mediums Mediums

MSA EA Mac Conkey PEA SS XLD Blood Agar Cetrimide Agar

The indicated bacterias are inoculated to some mediums in this experiment. Then, according to properties of
mediums, changes of mediums are observed. Also, changes of mediums give informations about properties of
bacterias. Thus, unknown bacterias are predicted.
 RESULTS
For Experiment 1: Observing of Alpha hemolysis, Beta hemolysis and Gama hemolysis with Blood Agar
Image 1: Blood Agar Inoculation
For Experiment 2: Inoculation Selective Media For Gram Positive Bacteria ( MSA or PEA)
Image 2: MSA Inoculation
Sheep blood agar is used to found hemolytic
colonies. Enterococcus faecalis do not use
hemoglobin in the sheep blood. So, changes of
zone are not observed on region of inoculation.
Non-hemolytic (gamma-hemolytic) colonies of
Enterococcus faecalis on sheep blood agar.
The hemoglobin in the blood agar is used by
Staphylococcus aureus. So, The white zone is
observed around colonies of Staphylococcus
aureus. On blood agar plates, colonies of
Staphylococcus aureus are frequently surrounded
by zones of clear beta-hemolysis.
The hemoglobin in the blood agar is used by X
bacteria. So, The green zone is observed around
colonies of X bacteria. On blood agar plates,
colonies of X bacteria are frequently surrounded
by zones of clear alfa-hemolysis.
While MSA supported the development of
halotolerant microorganisms such as
Staphylococcus species with a salt (NaCl) ratio of
7.5%; it prevents the growth of other bacteria that
can not tolerate the presence of salt in this
concentration. It also contains mannitol as a
carbon source and phenol red as a pH indicator.
This indicator is yellow if pH is below 6.6 and red if
pH is above 8. Many colonies are observed on the
types of Staphylococcus epidermidis and
Staphylococcus aureus. Also, these colonies have
pink color and yellow zone. But, the colonies of E.
coli and X bacteria are same with Staphylococcus
epidermidis and Staphylococcus aureus. Normally,
E.coli is not observed in the MSA. We can say that,
this petri dish is contaminated. The reason of
contamination will explain in the discussion. The X
bacteria is a unknown bacteria and this bacteria
can not occur colonies in the MSA. Thus,
Staphylococcus epidermidis and Staphylococcus
aureus are gram positive bacterias. Others are
gram negative bacterias.
For Experiment 3: Inoculation Selective Media For Gram Negative Bacteria ( EA )
Image 3: EA Inoculation
For Experiment 3: Inoculation Selective Media For Gram Negative Bacteria ( MAC )
Image 4: MAC Inoculation
The sodium sulphite and fucsin in the endoagar
composition suppress the growth of Gram positive
bacteria. Lactose fermentable (lactose-positive)
bacteria form lactose acid and aldehyde at the end
of the required incubation period. The resulting
aldehyde liberates the fucsin in the fucsin-sulphite
compound so that the colony becomes red in
color. In lactose-fermentable (lactose-positive)
bacteria this reaction occurs very strongly and the
fuksin crystals in the colony ensure that the colony
color is metallic bright green. Lactose-free (lactose-
negative) bacteria form colorless colonies in the
medium. In this experiment, E. coli and X bacteria
are observed metalic green colony color. Thus, E.
coli and X bacteria either gram negative or lactose
positive. But, the colonies are not observed on
Staphylococcus aureus. We can say that,
Staphylococcus aureus is either gram positive
bacteria absolutely or lactose-negative bacteria.
Mac Conkey Agar suppresses the growth of Gram
positive bacteria with bile salts and crystal violet
contained, Gram negative bacteria can easily
reproduce. Neutral red added to the medium as a
pH indicator indicates whether lactose is used as a
carbon source. This indicator is red when pH is
below 6.8 and yellow when pH is above 8. At the
end of the required incubation period, lactose-free
fermentable (lactose-negative) bacteria are raised
to pH by using the peptones in the medium as
energy sources and thus form colorless colonies.
Colonies of lactose-positive bacteria are red
because lactose fermentation reduces the pH of
the resulting acidic products below 6.8. The same
colonies are observed in E. coli, X bacterias and S.
aureus. S. aureus bacteria is known gram positive
and the colonies of this bacteria should not be
observed. In this situation, this petri dish is
contaminated. Also, E. coli bacteria is gram
negative and lactose positive because of the color
of background is pink. The about X bacteria can not
commet. The reason of contamination will explain
in discussion.
For Experiment 4: Inoculation Selective Media for Pathogenic Enteric Bacteria ( SS Agar )

Brilliant green, bile salts (even salts), sodium

thiosulphate and citrates (sodium and ferric
citrate) in the SS composition inhibit the
development of Gram positive bacteria (selective
nutrient). SS Agar contains lactose as a carbon
source. Neutral red added to the medium as pH
indicator indicates whether lactose is used. This
indicator is red when pH is below 6.8 and yellow
when pH is above 8. E. coli and X bacteria are not
use lactose as a carbon source. Thus, the ph is
acidic because of this two bacteria and pink zones
are occured. Sallmonella uses lactose as a carbon
source. Thus, the pH is alkaline and yellow zones
are occured. The Salmonella is lactose positive. The
E. coli and X bacteria are lactose negative.

Image 5: SS Agar Inoculation

For Experiment 4: Inoculation Selective Media for Pathogenic Enteric Bacteria ( XLD Agar )

Sodium deoxycholate, also found in XLD, inhibits

the growth of Gram positive bacteria. This medium
include xylose, lactose and sucrose as carbon
sources; The pH indicator contains phenol red
(phenol red). This indicator is yellow if pH is below
6.6 and red if pH is above 8. The pH of the medium
is lowered as xylose and / or lactose and / or
sucrose fermentable microorganisms cause
fermentation of the resulting acidic products. E.
coli and X bacteria have yellow zone. This situation
shows that E. coli and X bacteria use lactose or
xylose or sucrose. But, Salmonella has red zone
and pink zone has alkaline pH. In this situation,
Salmonella is gram negative and does not use
lactose or xylose or sucrose as a carbon source.

Image 6: XLD Agar Inoculation

For Experiment 5: Inoculation on Selective Medium for P. aeruginosa (Cetrimide Agar+Nalidixic Acid)

Pseudomonas aeruginosa grows well on all

normal laboratory media but specific isolation of
the organism, fromenvironmental ite sor from
human, animal or plant sources, is best carried
out on a medium, which contains a selectiveagent
and also constituents to enhance pigment
production. Most selective media depend upon
the intrinsic resistanceof the species to various
antibacterial agents. Cetrimide inhibits the growth
of many microorganisms whilst allowing
Pseudomonas aeruginosa to develop typical
colonies. Addition of nalidixic acid can aid in
inhibiting the growth of accompanying flora. This
petri dish shows that only there are colonies of
Pseudomonas species.

Image 7: Cetrimide Agar+NA Inoculation

Pseudomonas aeruginosa grows well on all

normal laboratory media but specific isolation
of the organism, fromenvironmental ite sor
from human, animal or plant sources, is best
carried out on a medium, which contains a
selectiveagent and also constituents to enhance
pigment production. Most selective media
depend upon the intrinsic resistanceof the
species to various antibacterial agents.
Cetrimide inhibits the growth of many
microorganisms whilst allowing Pseudomonas
aeruginosa to develop typical colonies. Addition
of nalidixic acid can aid in inhibiting the growth
of accompanying flora. This petri dish shows
that only there are colonies of Pseudomonas
species.

Image 8: Cetrimide Agar+NA Inoculation

For Experiment 5: Inoculation on Selective Medium for P. aeruginosa (Cetrimide Agar)

Cetrimide Agar and is used as a selective

medium for the isolation of Pseudomonas
aeruginosa from pharmaceutical products. This
medium is also used for microbial limit testing
for non-sterile products. This medium is also
used for determining the ability of an organism
to produce fluorescein and pyocyanin.
Cetrimide is incorporated in the medium to
inhibit bacteria other than Pseudomonas
aeruginosa. This compound a cationic
detergent acts as a quaternary ammonium
compound, which causes nitrogen and
phosphorus to be released from bacterial cells
other than Pseudomonas aeruginosa. But, E.
coli and X bacteria live in the this agar. This
situation shows that experiment is
contaminated. The reason of this situation will
Image 9: Cetrimide Agar
explain in discussion.

Cetrimide Agar and is used as a selective

medium for the isolation of Pseudomonas putida
from pharmaceutical products. This petri dish
shows that Salmonella does not live in the agar,
only Pseudomonas species live.

Image 10: Cetrimide Agar

 Drawings of Results
Image 1: Blood Agar Inoculation Image 6: XLD Agar Inoculation

Image 2: MSA Inoculation Image 7: Cetrimide Agar+NA Inoculation

Image 3: EA Inoculation Image 8: Cetrimide Agar+NA Inoculation

Image 4: MAC Inoculation Image 9: Cetrimide Agar

Image 5: SS Agar Inoculation Image 10: Cetrimide Agar

 DISCUSSION

The identification of higher plants and animals involves the observations of the structural differences, both
internal and external, which exist among them. Most of these structural differences are visible to naked eye.
Even the identification of microscopic plants and animals involves the observations of their structural
differences under a microscope. Such identification based on structural differences is not possible in the case
of bacteria, because structural differences, which may differ from one species of bacteria to the other, are not
discernible even under a microscope. Because the structural differences in shape, size and arrangement are
only helpful in the process of identification, there are many species of bacteria having similar shape, size and
arrangement. Therefore, ultimately, the identification of bacteria is mostly based on the differences in their
biochemical activities. There are many biochemical tests for the identification of bacteria. We will talk about
the mechanics of these tests, their consequences, and the mistakes in their execution. The survival and
continued growth of microorganisms depend on an adequate supply of nutrients and a favorable growth
environment. For survival, most microbes must use soluble low-molecular-weight substances that are
frequently derived from the enzymatic degradation of complex nutrients. In this experiment, blood agar is
used to observing hemolysis in bacteria types. The mannitol salt agar is used to observing salt resistant in
bacteria types. The bacterias are choosed according to gram negative or positive and lactose positive or
negative properties in MCA, EA, SS and XLD. Only Pseudomonas species are choosen in Cetrimide agar and
Nalidixic acid. Some experiments are wrongly maked such as experiment 2. Staphylococcus epidermidis and
Staphylococcus aureus are gram positive bacterias. Although, E. coli and X bacteria are gram negative
bacterias, these bacterias have to colonies. We say that, experiment is contaminated. The reason of
contamination, inoculation loop is not cleared enoughly. So, some bacterias remain on the inoculation loop.
Thus, wrongly positive results are observed. The one of wrongly maked experiment is experiment 3. Mac
Conkey Agar suppresses the growth of Gram positive bacteria. But, S. aureus has colonies. The this situation
shows contamination of experiment. The reason of this contamination is lack of sterilization. The same
situation exists in experiment 5.

RESOURCES

https://www.alice.cnptia.embrapa.br/bitstream/doc/1055907/1/276891388061PB.pdf
https://www.easynotecards.com/notecard_set/80649
https://www.scribd.com/document/216995056/Cetrimide-Agar-Base