Transcript Selective & differential

NAS
Lecturer: Mr. M. Zivuku
LECTURE 4
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Objectives
By the end of this unit, you should be able to;
• Explain the cultivation of bacteria in terms of in vivo and
in vitro.
• Describe why is it important to cultivate microorganisms.
• Describe and explain the different types of culture media
(Solid, semisolid, liquid, biphasic).
• Define the terms (i) selective media ,(ii) differential
media.
• list the composition of each of these culture media (i) and
(ii) above and explain why they are selective/differential.
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Cultivation of Bacteria
The process of growing microorganisms in
culture by:
– Taking bacteria from an infection site by
specimen collection - in vivo
– Growing bacteria in the artificial environment
of the laboratory - in vitro
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Why cultivate bacteria?
• Obtain definitive identification and
characterization
• Grow and isolate all bacteria present in an
infection
• Determine which bacteria is most likely
causing infection
• Determine which bacteria is likely a
contaminant or colonizer
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Types of Culture Media
-Solid, semisolid, liquid, biphasic
-Simple media, special media
(enriched, selective, enrichment,
indicator/ differential, transport)
- Aerobic and anaerobic media
-Cell culture for obligate intracellular
bacteria (e.g., Chlamydia spp)
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Biphasic Culture Medium
Agar slope
(solid medium)
Broth
(liquid medium)
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Selective and Differential Media
SelectiveThese are culture media in which chemical
substances are added to prevent growth one
group of bacteria without affecting the growth
of the other bacteria e.g. crystal violet will
inhibit the growth all G+ve organism
Example: Mac Conkey agar, Crystal violet
agar
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Selective Media
• Example:
• Bismuth sulfite for Salmonella typhi
(inhibits gram-positive and most gramnegative intestinal bacteria)
• It use glucose as a primary source of carbon
• Bismuth will stop G+ve growth
• Selectivity, utilize ferrous sulphate and
convert to Hydrogen sulphide
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Composition of Bismuth sulfite
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Bismuth Sulfate Bi2(SO3)3
Pancreatic digest of casein
Pancreatic digest of animal tissue
Beef extract
Glucose
Dibasic sodium phosphate
Ferrous sulphate.7 water
pH adjusted 7.7 at 25C
-1.6%
-1.0%
-1.0%
-1.0%
-1.0%
-0.8%
-0.06%
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Differential
•These are media to which dyes or other
substances are added to differentiate
microorganism. Such changes may include
color, pH , haemolysis, coagulation,
hydrolysis or fermentation –
•Example: Blood agar plates for
Streptococcus pyogenes
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Selective and differential Media
Differential
• Blood agar plates for Streptococcus pyogenes
Type of hemolysis reaction, identification of
Identification S. pyogenes
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Selective and differential
Selective & differential
•Mannitol salt agar
• Selective medium for pathogenic for
Staphylococcus aureus isolation in clinical
samples and biological and pharmaceutical
products.
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Composition
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TYPICAL FORMULA (g/l)
Beef Extract ……………………. 1.0
Peptospecial …………………..10.0
Sodium Chloride ……………..75.0
Mannitol …………………………10.0
Phenol Red …………………… 0.025
Agar ………………………………15.0
Final pH = 7.4 ± 0.2 at 25°C.
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Mannitol salt agar
• The high concentration of sodium chloride
(7,5%) generally inhibits the growth of
other bacteria.
• Fermentation of the mannitol causes an
acidification of the medium and a colour
change from red to yellow.
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Mannitol salt agar
• The coagulase positive staphylococci
cultivate with yellow colonies, surrounded
by a yellow zone.
• The coagulase negative staphylococci grow
less luxuriantly forming small red-purple
colonies.
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Principle of MSA
Summary
• This tests for the bacteria’s ability to
tolerate 7% salt concentration and ferment
mannitol. The media is selective because it
selects for salt tolerant bacteria. The media
is also differential because it differentiates
the salt tolerant organisms on their ability to
ferment mannitol
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Solid, Semi-solid and Fluid
culture media
• Solid Culture Media
• Media are solidified by incorporating a
gelling agent such as agar or gelatin
• Agar- a pollysacharide extract obtained
from seaweed and is commonly used to
solidify culture media because of high
gelling strength, its setting temp 32-39 Deg
C and melting temp 90-95Deg C
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Solid Media
• Solid media are used mainly in Petri dishes
as plate cultures.
• Also in bottles or tubes as stab (deeps) or
slope cultures
• NB
• The purpose of culturing on a solid medium
is principally to isolate discrete colonies of
each organism present in the specimen.
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Semi Solid Culture Media
• This form of culture medium is prepared by
adding a small amount of agar (0.4-0.5%) to
a fluid medium
• Semi solid are used mainly as transport
media and for motility and biochemical
tests
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Fluid culture Media
• Fluid media are most commonly used as
enrichment where organisms are likely to be
few e.g blood culture
• Some organisms produce a surface growth
on the in which they are growing e.g Vibrio
cholerae when grown in alkaline peptone
water
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