Media Preparation and Sterilization

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Media Preparation and Sterilization
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A medium is sterilized (living organisms removed)
before usage in the lab.Sterilization methods
include autoclaving, dry-heat, filtration, UV
exposure and ethylene oxide.Culture Is part
of specimen grown in culture media.Culture
Media is a medium (liquid or solid) that
contains nutrients to grow bacteria in vitro.
Because sometimes we cannot identify with
microscopical examination directly, and sometimes
we do culture for antibiotic sensitivity testing.
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Medium is a nutrient blend used to support
microbial growth.There are three physical forms
of media, broth, solid, and semisolid.Solid
media are more versatile in their
usage. Promote surface growth Used to isolate
pure cultures Ideal for culture
storage Helpful in the observation of
biochemical reactions Used to make slants,
deeps, and plates (named by medium)
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Properties of Media

• Support the growth of the bacteria.
• Should be nutritive (contains the required amount
  of nutrients).
• Suitable pH (neutral to slightly alkaline
  7.3-7.4).
• Suitable temperature, and suitable atmosphere.
  (Bacteria grow at 370C)
• Note media are sterilized by autoclaving at
  1210C and 1.02 atmosphere (15 p.s.i.) for 15-20
  minutes. With the autoclave, all bacteria, fungi,
  viruses, and spores are destroyed. Some media
  cant be sterilized by autoclaving because they
  contain eggs or carbohydrates .

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Forms of culture

• Solid (agar) Is Broth plus agar (seaweed).
• Are prepared by adding a solidifying agent
  (agar 1.5 -3).Prepared mainly in Petri dishes,
  but also in tubes and slopes. After growth the
  bacterial colonies are visible. e.g. blood agar,
  chocolate agar, MacConkey agar.
• Semisolid agar (soft agar) Contains small
  amounts of agar (0.5-0.7). Used to check for
  motility and also used as a transport media for
  fragile organisms. Can have semisolid agar in
  Petri dishes or in tubes. In tubes it is usually
  slanted to increase surface area.

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Liquid (Broth) Mostly used for biochemical tests
(blood culture, Broth culture). Growth of
bacteria is shown by turbidity in medium. e.g.
Nutrient broth, Selenite F broth, alkaline
peptone water.Properties of agarSome what
like gelatin.It melts at 850C and solidifies at
40-320C.Comes as sold powder and then you add
water to it.
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Types of Culture Media

• Simple (basal, ordinary) Culture Media are
  media that contain the basic nutrients (growth
  factors) that support the growth of bacteria
  without special nutrients, and they are used as
  basis of enriched media. E.g. Nutrient broth,
  nutrient agar, peptone water. They are for the
  growth of non-fastidious organisms like E. coli.

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Enriched Culture Media are media that are
enriched with Whole blood e.g. blood agar. Lysed
blood (heated to 85C) e.g. Chocolate
agar.Selective Media it is a media, which
contains substances that prevent or slow the
growth of microorganisms other than the bacteria
for which the media is prepared for. For
exampleEMB (Eosin Methylene blue) enteric
isolation media
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Differential Media (indicators) Contains
indicators, dyes, etc, to differentiate
microorganisms. E.g. MacConkey agar, which
contains neutral red (pH indicator) and is used
to differentiate lactose fermenter and
non-lactose fermenter. (E.g. E. coli and
Salmonella).
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Common media used in Microbiology Laboratory

• Chocolate Agar blood agar prepared by heating
  blood to 85C until medium becomes brown or
  chocolate in color heating the blood releases
  broth X and V growth factors and also destroys
  the inhibitors of V factor. These factors are
  required for the growth of most species of
  Haemophilus and also Neisseria gonorrhoear.
• MacConkey Agar an inhibitory and differential
  medium used to distinguish lactose- fermenting
  Gram- negative organism from non fermentation.
  Crystal violet, bile salts and neutral red are
  inhibitor agent. neutral red is the PH indicator.

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Mannitol Salt Agar ( MSA ) for selective
isolation for coagulase positive,
mannitol-fermenting staphylococcus. Mannitol
fermentation by pathogenic staphylococci is
indicated by a yellow halo surrounding the
colonies.Sodium chloride is the inhibitor
agent.Phenol red is the PH indicator.Mueller
Hinton Agar rich medium that support the growth
of most microorganism. It is commonly used for
antibiotic susceptibility testing disk diffusion
antibiotic susceptibility antibiotic serum level
measurements MBC determination.
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Salmonella Shigella ( SS ) Agar isolation and
differential medium for pathogenic Gram-negative
bacilli in particular, Salmonella and Shigella.
Inhibitor for Coliforms.Triple Sugar Iron Agar
(TSI) this a key medium for use in beginning
the identification of a Gram- negative bacilli of
the enteric group. It contains glucose (0.1 ),
Lactose (1), sucrose(1). And peptone (2) as
nutritional sources. Sodium Thiosulfate serves as
the electron receptor for reduction of sulfur and
production of H2S. Detects fermentation of
sucrose, lactose, glucose, as well as production
of hydrogen sulfide and /or gas . Phenol red is
the PH indicator ferric ammonium citrate is H2S
indicator.
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SINGLE MEDIA / MULTIPLE TESTS
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SINGLE MEDIA / MULTIPLE TESTS

• Several media are designed to yield more than one
  biochemical reaction. Among the more commonly
  used media in this category are
• SIM medium.
• Kliger's Iron agar (KIA).
• Triple Sugar Iron agar (TSIA).
• Lysine Iron agar (LIA).
• Motility Indole Ornithine (MIO) medium.

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SIM medium

• INGREDIENTS
• 0.4 agar ( semisolid).
• Peptone ( which rich in treptophan amino acid ).
• Sodium thiosulfate
• Ferrous ammonium sulfate. H2S INDICATOR

• The ingredients in SIM Medium enable the
  determination of three activities by which
  enteric bacteria can be differentiated.
• Sodium thiosulfate and ferrous ammonium sulfate
  for indication of hydrogen sulfide production.
  The ferrous ammonium sulfate reacts with H2S gas
  to produce ferrous sulfide, a black precipitate.
• The peptone is rich in tryptophan, which is
  attacked by certain microorganisms resulting in
  the production of indole. The indole is detected
  by the addition of Kovacs reagent.
• Motility detection is possible due to the
  semisolid nature of the medium growth radiating
  out from the central stab line indicates that the
  test
• organism is motile.

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Indole negative (Kovac's reagent did not turn
red) and hydrogen sulfide negative (agar did not
turn black).
SIM medium
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If indole is produced from the breakdown of the
amino acid tryptophan, the Kovac's reagent, when
added, will turn red.
SIM medium
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Indole negative (Kovac's reagent did not turn
red) and hydrogen sulfide positive (agar turned
black)
SIM medium
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Kligler's Iron agar (KIA)Triple Sugar Iron agar
(TSI)

• INGREDIENTS
• Enzymatic Digest of Casein.
• Enzymatic Digest of Animal Tissue.
• Yeast Enriched Peptone.
• Dextrose...................0.1 .
• Lactose......................1.0 .
• Sucrose......................1.0 . NOT PRESENT
  IN KLIGlERs IRON AGAR
• Ferric Ammonium Citrate. AS H2S PRODUCTION
  INDICATOR
• Sodium Chloride.
• Sodium Thiosulfate.
• Phenol Red. AS PH INDICATOR
• Agar............................1.5 .
• Final pH 7.4 0.2 at 25C.

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PRINCIPLE

• Enzymatic Digest of Casein, Enzymatic Digest of
  Animal Tissue, and Yeast Enriched Peptone provide
  the nitrogen, carbon, and vitamins required for
  organism growth.
• Triple Sugar Iron Agar contains three
  carbohydrates, Dextrose, Lactose and Sucrose.
  When the carbohydrates are fermented, acid
  production is detected by the Phenol Red pH
  indicator.
• Sodium Thiosulfate is reduced to hydrogen
  sulfide, and hydrogen sulfide reacts with an iron
  salt yielding the typical black iron sulfide.
• Ferric Ammonium Citrate is the hydrogen sulfide
  (H2S) indicator.
• Sodium Chloride maintains the osmotic balance of
  the medium.
• Agar is the solidifying agent.

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Results

• An alkaline slant-acid butt (red/yellow)
  indicates fermentation of dextrose only.
• An acid slant-acid butt (yellow/yellow) indicates
  fermentation of dextrose, lactose and/or sucrose.
  
• An alkaline slant-alkaline butt (red/red)
  indicates dextrose ,lactose or/ sucrose were not
  fermented (non-fermenter).
• Cracks, splits, or bubbles in medium indicate gas
  production.
• A black precipitate in butt indicates hydrogen
  sulfide production.

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FIG. B The small amount of acid produced in the
slant of the tube during dextrose fermentation
oxidizes rapidly, causing the medium to remain
red or revert to an alkaline pH. In contrast, the
acid reaction (yellow) is maintained in the butt
of the tube because it is under lower oxygen
tension.
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Interpretation Symbol Results (slant/butt)
Glucose fermentation only Peptone catabolized K/A Red/yellow
Glucose and lactose and/or sucrose fermentation A/A Yellow/yellow
No fermentation Peptone catabolized K/K Red/red
No fermentation Peptone used aerobically K/NC Red/no color change
Glucose and lactose and/or sucrose fermentation Gas produced A/A,G Yellow/yellow with bubbles
Glucose fermentation only Gas produced K/A,G Red/yellow with bubbles
Glucose fermentation only Gas produced H2S produced K/A,G, H2S Red/yellow with bubbles and black precipitate
Glucose fermentation only H2S produced K/A, H2S Red/yellow with black precipitate
Glucose and lactose and/or sucrose fermentation H2S produced A/A, H2S Yellow/yellow with black precipitate
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Lysine Iron agar (LIA)

• INGREDIENTS
• Enzymatic Digest of Gelatin.
• Yeast Extract.
• Dextrose...............0.1 .
• L-Lysine...............1.0 .
• Ferric Ammonium Citrate. AS H2S INDICATOR
• Sodium Thiosulfate.
• Bromocresol Purple. AS PH INDICATOR
• Agar......1.5 .
• Final pH 6.7 0.2 at 25C.

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BROMOCRESOL PURPLE PH INDICATOR
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Lysine Iron agar (LIA)
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Motility Indole Ornithine (MIO) Medium

• INGREDIENTS
• Yeast Extract.
• Peptone.
• L-Ornithine.. 0.5.
• Dextrose ..............0.1.
• Agar . 0.4.
• Bromcresol Purple. PH INDICATOR
• Final pH 6.5 0.2 at 25C.

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PRINCIPLE

• MIO Medium is prepared to provides three
  differentiating tests in one culture tube
  motility, indole production, and ornithine
  decarboxylation. The principles of each are
  outlined below
• MOTILITY Organisms are stabbed into the
  semisolid medium using a straight wire. If the
  organisms are motile, they will migrate from the
  stab line by means of their flagella, producing
  turbidity or cloudiness throughout the medium.
  Non-motile organisms grow only along the stab
  line, leaving the surrounding medium clear.
• INDOLE Tryptophan is an ingredient contained in
  the medium by the inclusion of peptones. If the
  organism possesses the enzyme tryptophanase, with
  the addition of Kovacs reagent, a red color will
  form at the top of the medium if indole is
  present. This reaction occurs as a result of the
  indole reacting with the aldehyde to yield a
  quinone or quinone-type structure, resulting in a
  red color in the alcohol layer. A negative test
  results in no color change.

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PRINCIPLE continue.

• ORNITHINE The medium also tests for the presence
  of the enzyme ornithine decarboxylase by
  including L-ornithine in the agar. If the
  organism possesses the enzyme, it will be
  activated in an acid environment created by the
  initial fermentation of glucose. Once the amino
  acid is decarboxylated, the by-product diamine
  putrescine is produced. The result is a shift in
  pH to the alkaline range, turning the medium a
  dark purple. Organisms, which do not possess the
  enzyme, will stay in the acid range due to the
  fermentation, resulting in a yellow color in the
  medium.

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BROMOCRESOL PURPLE PH INDICATOR
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Motility Indole Ornithine (MIO) Medium