Cultivation of

microorganisms

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Media - the nutrient preparations that are used for culturing microorganisms

Properties of Media:
1.should be nutritive (contains the required amount of nutrients)- proteins, fats,
carbohydrates, or lipids, etc.
2.contain mineral salts – sulphate, phosphates, chlorides, carbonates of K, Ca, Mg
3.a proper moisture
4.a suitable pH – 7,2-7,4
5.accesory growth factors – tryptophan for Salmonella typhi or X & V factors for
Haemophilus influentzae
6.sterile (initially)

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Culture Media Consistency Classification
Three physical forms are used:

•liquid, or broth, media- − water-based solutions that do not solidify at

temperatures above freezing and that tend to flow freely when the container is
tilted (ex.: Nutrient broth, Peptone solution, etc.);
•semisolid media - used to determine the motility of bacteria and to localize a
reaction at a specific site (ex.:; Cystine trypticase agar medium)
•solid media
- provide a firm surface on which cells can form discrete colonies
- usually used as: slants, stabs, petri dishes (ex.: Coagulated blood serum)

Culture Media Complexity Classification

1.Simple media – consists of only basic necessitie; used to culture non-fastidious
bacteria (agar plate; nutrient broth; peptone water)
2.Complex media - for nutritionally more exigent bacteria
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 Bacteriological Media Application
Classification

1. Basic media – for the general cultivation of

bacteria (agar plate, bouillon, peptone water)
2. Storage media - medium in which bacteria are
stored in "stock culture" (Yeast extract mannitol
agar medium)
3. Enrichment media – used to select a desired
organism from a specimen with mixed flora
(addition of extracts of plant or animal tissues to
Nutrient broth or Nutrient agar media provides
additional nutrients and the media start favouring
the growth of fastidious heterotrophic bacteria)

Nutrient broth
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4. Selective and differential media
• Selective media – are growth media that contains substances that inhibit the growth of
unwanted organisms but permit the growth of the desired organism; uses certain dyes,
high salt concentration, pH or antibiotics
• Differential media - supports the growth of several types of microorganisms, but it is
designed to highlight differences among these microorganisms; contains a combination
of nutrient and pH indicators to visually differentiate bacteria that grow on or in it
• Examples:
 Mannitol salts agar (selects against non-skin flora; for the selection of the S.aureus;
mannitol fermentation = yellow)

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MacConkey agar (selects against gram-positives; for the selection of the
Enterobacteriaceae and related gram-negative bacilli;) (lactose fermentation = red)

•Salmonella-Shigella medium (a high selective medium; inhibit the growth of

most coliform organisms; permit the growth of Salmonella and Shigella; lactose
fermentation = red)

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 Bile Esculin Agar (used to identify
members of the genus Enterococcus (E
faecalis and E. Faecium); tests the ability
of organisms to hydrolyze esculin in the
presence of bile = brown to black)

Biochemical Test Media – used to isolate and identify bacteria from clinical
specimens; contain a sugar ( glucose, lactose, sucrose, etc.) or another biochemical
substance such as: indole, methyl red, citrate (Simmon’s Citrate Agar), urea, and a
colour indicator. Ex.: TSI (triple sugar iron), SIM (Sulfur Indole Motility),etc.

urease test
TSI SIM 7
 Anaerobic media – are nutritional base schaedler blood BHI
media used for the cultivation of agar
anaerobic bacteria (will grow only in the
absence of O2) from clinical specimens
( liqiuds or solids- schaedler blood agar)

 Media for antibiotic susceptibility

testing - Mueller-Hinton agar

 Transport medium – is a special

purpose media that contains balanced
salts to protect the specimen from pH
changes and keeps the swab moist while
in transit to the laboratory for culturing Amies→
( ex. Cary- Blair transport medium for the
enteric pathogens). Stuart→

Cary- Blair→ 8
 Isolation of bacteria in pure culture
Common Methods of isolation of pure culture
Liquid media

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Solid media
• Streak plate technique
• Inoculation -on the surface of a solid medium in a Petri dish
• Rational of procedure - to physically separate the various cells one from another by spreading
them over solid surface
• The bacterial suspension is streaked on an agar plate with the aid of an inoculating loop.
• As the streaking goes on, fewer and fewer cells are left on the loop and finally single cells
attach to the surface of the medium.

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Inoculating a deep

Sterilize the needle (until red hot), wait a few

seconds and than pick your sample, stab the
needle in the middle of the deep and remove it
through the same stab.

 Inoculating an agar slant

Sterilize the loop (until red hot) and wait a few seconds. Touch the loop to an isolated colony and
carefully place it into the sterile slant tube. Using a zig-zag motion, move the inoculating loop
along the SURFACE of the sterile agar slant from the bottom of the slant up to the top so that a
maximum surface area is exposed to the bacterial culture.

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 anaerobic jar
Isolating anaerobic bacteria
Solid media

•the anaerobic jar - It is a medium-sized glass or

plastic jar with a tightly fitting lid.
It can be set up by two methods:

1.a commercially available hydrogen and carbon

dioxide generator envelope (GasPak) is placed in
the jar along with the culture plates the generator is
activated with water
• Oxygen within the jar and the hydrogen that is generated are converted to water in
the presence of the catalyst, thus producing anaerobic conditions.
• Carbon dioxide - required for growth by some anaerobes and stimulates the growth
of others.
2. evacuation and replacement
• air is evacuated from the sealed jar containing the culture plates and is replaced with
an oxygen-free mixture of 80 percent nitrogen, 10 percent hydrogen, and 10 percent
carbon dioxide.
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• Liquid media

• Heat a broth tube to boiling to drive off all oxygen (10-20 min)
• Cool it to 45 to 50o C in water;
• Add the sample that may contain anaerobe;
• Use your loop- stir a bit to disperse the bacteria without getting air back into the tube.
• 2 ml of paraffin oil is added to seal the medium from the air
• Incubate ;

Aspects of bacterial growth

Liquid media
• Growth of bacteria is shown by turbidity in medium.

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Solid media
Bacteria grow on solid media as colonies.
A colony - a visible mass of microorganisms all originating from a single mother cell
Basic elements - for all colonies:
•Surface – smooth (S), glistening (G), rough (R), mucoid (M), etc.
•Form – the basic shape of the colony- circular, filamentous,etc.
•Elevation - the cross sectional shape of the colony
•Margin - the magnified shape of the edge of the colony
•Opacity - transparent (clear), opaque, translucent , etc
•Pigmentation – white ( S.epidermidis), buff, red, purple, etc.
•Consistency
•Odor
•Mobility

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 Cultivation of viruses

• can replicate only in receptive living cells

• these are provided by cell cultures, embrionated eggs or animals.

• Cell cultures: in vitro biological system formed by isolated viable cells that
maintain their capability to multiply;
• to retain the cells viability nutrients, energy source, aseptic conditions, proper
temperature, pH must be provided.
 primary cultures – derived from freshly removed animal/human,
normal/tumor tissue, obtained by dispersing cells; cells in primary cultures are
unable to grow for more than a few passages;
 diploid cell cultures (secondary cell cultures) – derived from embryos – cells
are viable for about 50-60 passages
 continuous cell lines – derived from malignant tissues, capable for indefinite
growth o ex. HeLa, Vero, Hep2, MacCoy, etc.

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Detection of virus-infected cells:

- cytopathic effect: cell lysis, necrosis, inclusion formation, giant cell

formation,
cytoplasmic vacuolization
- adsorbtion of eryhrocytes to infected cells, called hemadsorbtion, due to
the expression of a viral protein (hemagglutinin – influenza virus) on the
membrane of infected cells; this becomes positive before cytopathic effects
become visible
- detection of virus-specific nucleic acid

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