this slide will help you to understand the behavior of different types of bacteria in different culture media. its is made with an exmaple experiment which can provide better understadng. selective, differential and enriched media is given with detailed description in the example.

1. Microbiological
Culture Media

2. Group B-2
Made by Sadia Haque
3rd professional year,
Pharm-D, University of
Karachi.

3. 1.Microbiological culture
It is a method that involves the growth of bacteria or other
microbial organisms by allowing them to feasibly reproduce
in a pre-determined culture media which has all the
conditions under laboratory control. In other words, it
involves the cultivation of bacteria. Cultivation is a process in
which various microbes are grown in culture from the site of
infection (in vivo) on artificial media in the laboratory (in vitro)
to aid in the diagnosis and treatment of disease.

4. 2.Culture media
All the studies regarding microorganisms depend upon its
sufficient and healthy growth in the laboratory.
Different types of bacterias with different nature require
suitable culture media to grow feasibly.
A culture media can be prepared as a solid or a liquid
preparation which can either be used for the growth of
transportation and storage of the microorganism.
The quality of a suitable culture media is that it contains all the
required nutrition for bacterial growth.

5. 3.Types Of Growth Media
1. Liquid media
2. Semi-Solid media.
3. Solid media (liquefiable)
4. Solid media (non-liquifiable)
Growth media exists in the following states.

6. 3.1.Liquid broth
Liquid broth/ liquid media does not have any solidifying
agent that is why it remains in a liquid state. Liquid broths
change its appearance by turning from clear to turbid or
cloudy, which is an indication of active bacterial growth. In
this process, desired bacteria are suspended in a liquid
medium with all the required nutrients, for example, Luria
Broth, in an upright flask.

7. 3.2.Semi-Solid media
Semi-solid media is also known as Sulphur
Indole Motility Media SIMM. It has only 0.3
to 0.5 % of Agar/Solidifying agent. This type
of media is very useful in demonstrating
bacterial motility and separating motile and
non-motile strains of bacteria.

8. 3.3&4.Solid Media
The other way of growing microbiological cultures is by
using Petri dishes of different sizes. Solid agar is made
from a few ingredients that include water, nutrients, and
agarose gel, which is a solidifying agent. The petri dish
which contains the agar is referred to as an agar plate.
Agar plates contain a thin layer of agar-based growth
medium. To start the growth of the bacteria the agar-
based growth medium is inoculated with the desired
bacteria. The NON-LIQUEFIABLE solid media is made
of plant or animal dried extracts.

9. 4.Classification Of Media:
1. Selective Media.
2. Differential Media.
3. Enriched/Enrichment Media.
4. Transport Media.
5. Enumeration Media
6. Assay Media.

10. 4.1. Selective Media
This media has certain substances that inhibit the
growth of all but a few bacteria and at the same
time facilitate isolation of certain bacteria.
Few examples of selective media include:
● Thiosulfate citrate bile salt sucrose agar
(TCBS)
● Deoxycholate Citrate Agar (DCA)

11. 4.2.Differential media
Differential or indicator media is used to distinguish
between microorganisms that are growing on the same
media, by their growth nature and characteristics. Some
bacterias can be differentiated by pH indicator for example
macconkey Agar can be used to differentiate between
lactose fermenter and non-lactose fermenter. Other
examples include;
● Eosin Methylene Blue (EMB)
● Mannitol Salt Agar (MSA)

12. 4.3.Enriched and Enrichment Media
ENRICHED MEDIA is the kind of solid media that supports and facilitates
the growth of certain fastidious bacteria. Enriched media ingredients
contain blood, serum and egg so that all the nutritional requirements are
satisfied for more exacting and more fastidious bacteria. Few examples of
enriched media includes
● Blood Agar
● Chocolate Agar
While ENRICHMENT MEDIA is the kind of liquid media which isolates and
facilitates the certain type of bacteria but inhibits others. Selenite f broth is
an enrichment media that is most commonly used to isolate Salmonella
typhi.

13. 4.4. Transport Media
Transport media is specifically
designed for maintaining the viability of
certain delicate organisms in a clinical
specimen while they are transported to
the laboratory.

14. 4.5.Enumeration Media
It is the kind of media that is used to enumerate or count
the number of organisms in different food products, soil
and other samples.

15. 4.6.Assay Media
This is the kind of media that is used as an assay of
vitamins, amino acids and antibiotics. The most commonly
used assay media is antibiotic assay media which helps to
determine the potency of the antibiotic.

16. 5.Preservation Of Culture And Methods
To Preserve Culture.
Microbiological culture is preserved for different reasons, which
includes study purposes, different types of research by scientists
to get a better understanding of these organisms and also for
foods and beverages.

17. 6.Disposal Of Culture
The whole culture must be heated to destroy microorganisms
before disposal. An autoclave or cooker can be used, in
conjunction with autoclavable bags. The caps of all screw-
topped bottles must be loosened before cultures and media
are sterilised. Must make sure all the instructions for use of the
autoclave are followed in order to fulfil and maintain adequate
high temperatures for an extended enough time. Incineration
can be a reasonable alternative to autoclaving. In order to
prevent harmful fumes from creating any health hazard, a
purpose-built incinerator with a tall flue should be preferred.

18. Phenylethyl Alcohol Agar
PEA agar is a selective medium that is used for the isolation of
gram-positive Staphylococcusspecies andStreptococcus species
from clinical specimens or specimens that contain mixtures of
bacterial flora. Typically PEA agar is used to inhibit the common
contaminants such as Escherichia coli and Proteus species. PEA
agar may be prepared with and without 5% sheep blood
supplement. PEA agar with 5% sheep blood is used to isolate most
gram-positive and gram-negative anaerobes from enteric samples.
It is used to inhibit facultative gram-negative rods, preventing
Enterobacteriaceae from overgrowing the anaerobes and inhibiting
swarming of Proteus and Clostridium septicum . PEA agar is used
for purulent specimens and when mixed infections are suspected

19. Crystal Violet Agar
Crystal Violet Lactose Agar is used for the differentiation of pure cultures of
pathogenic from nonpathogenic strains of Staphylococci. The toxicity of
Staphylococci is estimated on the basis of their pigment production, haemolytic
and coagulating characteristic. Staphylococci produces different coloured growths
when cultured on Crystal Violet Agar. Haemolytic and coagulating strains
produced purple to violet colour whereas non-hemolytic and non-coagulating
strains produced white colonies after incubation. Crystal violet inhibits most of the
gram-positive organisms and is markedly inhibitory to Staphylococci. A fair
growth can be obtained at a 1: 300,000 concentration of the dye when the
medium is inoculated heavily. So, this medium is used for study of pure cultures
where a mass inoculation can be used rather than for primary isolation.

20. 7.5% Sodium Chloride Agar
7.5 per cent NaCl agar inhibits gram-negative organisms
which is why it is used to differentiate between gram-
negative and gram-positive microorganisms. It only allows
the growth of gram-positive organisms.

21. Mannitol salt agar
Mannitol salt agar or MSA is a commonly used selective and differential growth
medium in microbiology. It encourages the growth of a group of certain
bacteria while inhibiting the growth of others. This medium is important in
medical laboratories as one method of distinguishing pathogenic microbes in a
short period of time. It contains a high concentration (about 7.5–10%) of salt
(NaCl), making it selective for most Gram-negative and some Gram-positive
bacteria (Staphylococcus, Enterococcus and Micrococcaceae) since this level
of salt is inhibitory to most other bacteria. It is also a differential medium for
mannitol-fermenting staphylococci, containing carbohydrate mannitol and the
indicator phenol red, a pH indicator for detecting acid produced by mannitol-
fermenting staphylococci. Staphylococcus aureus produces yellow colonies
with yellow zones, whereas other coagulase-negative staphylococci produce
small pink or red colonies with no colour change to the medium.

22. Eosin methylene blue
Eosin methylene blue (EMB, also known as "Levine's
formulation") is a selective stain for Gram-negative bacteria. EMB
contains dyes that are toxic to Gram-positive bacteria. EMB is the
selective and differential medium for coliforms. It is a blend of two
stains, eosin and methylene blue in the ratio of 6:1. EMB is a
differential microbiological medium, which slightly inhibits the
growth of Gram-positive bacteria and provides a color indicator
distinguishing between organisms that ferment lactose (e.g., E.
coli) and those that do not (e.g., Salmonella, Shigella). Organisms
that ferment lactose display "nucleated colonies"—colonies with
dark centers.

23. MacConkey agar
MacConkey agar is an indicator, a selective and differential
culture medium for bacteria designed to selectively isolate
Gram-negative and enteric (normally found in the intestinal
tract) bacilli and differentiate them based on lactose
fermentation. The crystal violet and bile salts inhibit the
growth of Gram-positive organisms which allows for the
selection and isolation of gram-negative bacteria. Enteric
bacteria that have the ability to ferment lactose can be
detected using the carbohydrate lactose, and the pH indicator
neutral red.

24. Blood Agar
Blood agar is an enriched, bacterial growth medium. Fastidious
organisms, such as streptococci, do not grow well on ordinary
growth media. Blood agar is a type of growth medium (trypticase
soy agar enriched with 5% sheep blood) that encourages the
growth of bacteria, such as streptococci, that otherwise wouldn’t
grow. Certain bacterial species produce extracellular enzymes that
lyse red blood cells in the blood agar (hemolysis). These
hemolysins (exotoxin) radially diffuses outwards from the colony (or
colonies) causing complete or partial destruction of the red cells
(RBC) in the medium and complete denaturation of hemoglobin
within the cells to colorless products.

25. Type of medium Medium Bacterial Species Amount of Growth Appearance of Growth Appearance of Medium
Selective
Phenylethyl
alcohol
agar
E. coli (-) PEA inhibits the growth
of E. coli. Because it
disturbs its DNA
synthesize, so no growth
appears.
The medium remains
unchanged.
S. aureus (+++) Growth is exhibited with
red and opaque
colonies.
Medium shoes opaque
colonies on the
inoculated region.
E. faecalis (+++) Growth is exhibited with
opaque colonies.
Medium shoes opaque
colonies on the
inoculated region.
Crystal
violet agar
E. coli (-/+) Purple colonies The medium remains
unchanged in colour.
S. aureus (-) No colonies The medium remains
unchanged.
E. faecalis (-) No colonies The medium remains
unchanged.
7.5%
sodium
chloride agar
E. coli (-) No colonies The medium remains
unchanged.
S. aureus (+++) Yellow colonies with
yellow zones.
Medium changes its
colour to yellow.
S. epidermidis (+++) Pink colonies appears. The medium remains
unchanged in colour.

26. Differential/
Selective
Mannitol
salt agar
E. coli (-) No colonies. Medium remains
unchanged
E.aerogenes (-) No colonies. Medium remains
unchanged
S. aureus (+++) Small yellow colonies with
yellow zones.
It ferment mannitol &
medium changes its colour
from pink to yellow.
S. epidermidis (++) Colonies are colourless. Does not ferment mannitol,
medium remains pink in
colour.
MacConkey
agar
E. coli (+++) Flat, dry, pink colonies. Absence of medium and
pink halo appears around
the growth with ppt of bile
salts. Lactose fermented
E.aerogenes (++) Red colonies in prominent
amount.
Fewer bile salts ppt.
Lactose fermented.
S. typhimurium (+) Colourless or transparent
colonies.
Medium is unchanged and
lactose not fermented.
S. aureus (-) No colonies. Medium remains
unchanged.
Eosin–
methylene
blue agar
E. coli (+++) Green metallic sheen
colonies are formed.
Lactose is metabolized
forming green sheen.
E.aerogenes (+++) Large pink colonies with
blue center.
Lactose is metabolized.
S. typhimurium (+++) Rich growth of colourless to
grey colonies.
Lactose not fermented.
S. aureus (-/+) Colourless colonies are
formed.
Lactose not fermented.

27. Enriched Blood agar S. mitis (++) Dry grey colonies. Beta-hemolysis occurs
in medium causing
decolourization of
medium.
E. faecalis (++) Slight transparent to
grey colonies.
Gama-hemolysis
occurs medium remain
unchanged.
Streptococcus
var. Lancefield Group E
(++) Small colourless
colonies.
Alpha-hemolysis
occurs in medium
causing decolourization
of medium.

28. Result
Selective media:
Phenylethyl Alcohol Agar and 7.5 % NaCl Agar results to be
selective for gram-positive organisms i-e S.Aureus,
S.epidermidis and E-faecalis, while Crystal Violet Agar results
to be selective for Gram-Negative organisms i-e E-Coli.

29. Differential/Selective media:
Mannitol salt Agar results to be differential for mannitol fermentation
which is done by S.aureus, and its selective for gram-positive organisms,
while both McConkey and EMB Agar result to be differential for lactose
fermentation shown by E-Coli and E-aerogenes and selective for Gram-
Negative organisms.
Enrichment media:
Blood Agar results in facilitating the growth of all three fastidious
organisms and demonstrates all three kinds of haemolysis in the
medium.

30. Discussion
Selective medium: It is observed that PEA agar, Crystal Violet agar and
7.5% NaCl are all selective to different types of bacterial strains for
isolation purposes. PEA Agar is selective for gram-positive staphylococcus
and Streptococcus species that are taken from clinical specimens which
are impure. And 7.5% NaCl agar is also selective for the gram-positive
organism, which is why both PEA and 7.5% NaCl Agar inhibits the growth
of E-coli and facilitates the growth of staphylococcus aureus. While the
Crystal Violet Agar inhibits most of the gram-positive organisms being
selective for Gram-Negative, which is why it inhibits the growth of
staphylococcus aureus and facilitates E-coli.

31. Differential/Selective medium: Mannitol salt agar McConkey agar
and Eosin Methylene Blue Agar are all selective and differential both.
Mannitol salt Agar can differentiate between mannitol fermenting and
mannitol non-fermenting bacterias by indicating the pH change in the media.
While McConkey and EMB Agar differentiate between lactose fermenting
and non-fermenting bacteria. EMB shows nucleated colonies in case of
lactose fermenting bacteria which is why both E Coli and E-aerogenes both
being lactose fermenters in nature, show nucleated colonies. Mannitol salt
Agar is selective for gram-positive organisms which is why it facilitates the
growth of staphylococcus aureus and inhibits E-Coli. But McConkey and
EMB both being selective for gram-negative organisms facilitate the growth
of E-coli inhibiting staphylococcus aureus.

32. Enrichment media: blood Agar being enriched in nature provides
nourishment to the fastidious organisms. These organisms perform
haemolysis by lysing RBCs and causing decolourization of the medium.
Beta haemolysis occurs when there is complete decolourization of the
media around the colony which is done by Streptococcus mitis. While
Alpha haemolysis is performed by Streptococcus Var. Lancefield group E
( Streptococcus anginosus) which causes incomplete decolonization of
media. Gamma hemolysis/ No hemolysis is performed by E-faecalis
making no change to the colour of the medium.

33. References:
● Textbook of Microbiology & Immunology, Parija, Shubhash Chandra,
● Washington JA. Principles of Diagnosis. In: Baron S, editor. Medical microbiology.
● MacConkey AT (1905). "Lactose-Fermenting Bacteria in Faeces". J Hyg (Lond). 5
● Levine, M (1918). "Differentiation of B. coli and B. aerogenes on a simplified eosin-methylene blue
agar". J Infect Dis. 23: 43–47.
● Hans Günter Schlegel (1993). General Microbiology. Cambridge University. p. 459.
● Ryan KJ, Ray CG, eds. (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill.

34. Made by Sadia Haque
Thank you