PW6 - Microbiological culture media – classification, general presentation

1. Terminology:
Microbial culture = a population of microorganisms living in a culture medium
Growth of bacteria = increase in quantity of all cellular components, followed
by cell division
Bacterial division = usually, binary fission; other possibilities: budding,
sporulating, and splitting into fragments (Actynomycetes)
Generation time = the time needed by a cell of a bacterial population to double
itself
Population = all individuals belonging to one species, which are living in a
biotope
Clone = Population resulted from a single cell (Colony Forming Unit) by
vegetative multiplication
Strain = Microbial population resulted from a single isolate in pure culture
Microbiological culture media = combination of nutritive complex substances
which are able to support bacterial growth
Culturing = obtaining a microbial culture by seeding or inoculating the
microorganisms on microbiological culture media
Seeding = passing a quantity of a biological sample or of microorganisms
developed on a culture media on the surface of a non cellular solid medium or in a
non cellular liquid medium
Inoculation = introducing a quantity of microorganisms in a cellular medium:
cell culture, embryonated egg, experience animals

2. Classification of culture media

Cellular media (including living cells): cell cultures, embryonated eggs, experience
animals
Experience animals: white mouse, guinea pig, rat, rabbit etc. – used for:
growing microorganisms which can’t be cultivated on non-cellular media (Treponema
pallidum)
pathogenity tests
increasing the virulence of bacterial strains by repeating inoculation on animals:
Mycobacterium tuberculosis, Streptococcus pneumoniae, Klebsiella pneumoniae)
Choosing the experimental animal and the inoculation procedure is
depending on the pathogenity characters of the microorganism and on the
host innate susceptibility.
Experience animals are very expensive.
Embryonated egg – after 6-14 days of eggs incubation
- Specialized units to provide diagnosis and production dedicated eggs: sterile
and free of microorganisms
Steps:
- Ovoscopy
- Aseptic inoculation, in specific zones of the embryo, depending on the
microorganism: intramuscularly, into the amniotic cavity, chorialantoidian
membrane etc.)
 - Incubation at (37 0 C), ensuring the needed oxygen, hydration, incubation time
etc. (ex. Rickettssia, Chlamydia etc.)
Cell cultures:
organ cultures
dispersed cell cultures: primary cultures (enzymatic dispersion of the cells of a freshly
prelevated organ – 3-5 passages), cellular strains (homogeneous – 50-60 passages may
be used), cellular lines (stable), derived from normal or malignant cells, may be
subcultivated unlimited cf. recommended protocol
Ex. cellular lines derived from:
- Monkey kidney (Vero, BSC-1,BGMK), mouse (McCoy), hamster ovary
(CHO), uterus carcinoma (HeLa), larynx (Hep2) etc.
- different species prefer different lines (ex. Chlamydia species different lines)
- used for detecting toxins: ex. Vero line for Shigella shiga toxin and E. coli
O157 : H7 toxin, CHO line for V. cholerae and enterotoxigenic E. coli
Non-cellular media – cheaper or quite expensive, pending on the complexity, but less
expensive tha experience animals; special or routine media
Required qualities:
- sterility
- being growth suitable by their nutrients content
- adequate pH (usualy: 7,2 – 7,6)
- adequate osmotic pressure
- appropriate humidity for microbial growth
- appropriate ingredients for special media to sustain fastidious
microorganism’s growth etc.
Classification of non-cellular media:
Solid, semisolid, liquid
Simple, complex (provide the full range of complex growth factors)
Media for: transport, isolation, identification, preservation of bacteria
Hydrated or dehydrated (pulvis)
Special media: elective, selective, enrichment, enriched
3. Examples:
Solid media: simple nutritive agar, blood agar, BBL- A = Bromthymole Blau
Lactose Agar, Chapman, Mueller Hinton, Mac Conkey
Liquid media: alkaline peptone water, Mueller-Hinton broth, nutritive broth,
blood broth, selenit broth, thyoglycolat broth
Semisolid media: Cary-Blair, MIU = Motility Indole Urea , Stuart etc.
Simple media: agar base, nutrient broth etc.
Complex media: blood agar, chocolate agar, agar with X and V supplement,
Mueller-Hinton etc)
Transport media: Cary Blair, Amies, charcoal media etc.
Identification media: TSI = Triple Sugar Iron, MIU = Motility Indole Urea, API
galeries etc.
Preservation media: nutrient agar, WHO medium = World Health Organization
medium etc.
Special media:
- Elective media - best ingredients to support a specific bacteria growth:
Loeffler, with coagulated bovine serum for C. diphtheria
- Selective media – contain ingredients that will inhibit or prevent growth of other
bacteria than the targetted one; ex. blood agar with sodium azide – inhibits the
gram negatives, Chapman (Sodium chloride in high concentration inhibits
bacteria except staphylococcus), Mac Conkey, media with antibiotics supplement
etc.
- Enrichment media: enhance the growth of a few desired microorganisms among
large number of normal flora; they function concomitantly as elective and
selective media: selenit broth for Salmonella etc.
- Differential media – help to differentiate bacteria based on certain characters:
e.g. hemolysis on blood agar (on blood agar), capacity to use certain sugars as a
growth substrate (in this case they contain an indicator of this substrate
modification): AABTL (Agar Bromthymol Blue Lactose), McConkey agar,
ADCL (Agar Deoxycolate Citrate Lactose), or other characters MIU (Motility
Indol Ureea) etc.

Classification according to the physical characters:

- Dehydrated – we use distilled water, demineralized following the producer
recommendation in order to obtain the media that are used for cultivation in Petri
dishes
- Ready to use, in Petri dishes

Media presented in the classroom:

0
Blood agar: agar with blood added at 50 C; preserved in good condition for 4
weeks at + 4 0 C, in plastic bag

Chocolate agar = blood agar heated for 10 ‘at 80 0 C; delivering of the growth
factors from the erythrocytes; preserved in good condition for 4 weeks at + 4 0 C,
in plastic bag

Amies – agar, Sodium thyoglycolate, neutral pharmaceutical charcoal – charcoal

adsorbs the inhibitors – used for fastidious microorganisms – ex. Neisseria
gonorrhoeae; preserved in good condition for 12 months at + 4 0 C, in plastic bag

Selenite broth – enrichment medium for Salmonella spp.; selenite – toxic;

sterilized by boiling, not by autoclaving; preserved in good condition for 6
months at + 4 0 C, in plastic bag

Cary Blair – agar, Na thyioglycolate, anorganic salts; transport medium for Gram
negatives

Loewenstein-Jensen – mycobacteriology: salts, starch, malachit green, eggs

homogenate; screw caped tubes; coagulated in slopes, at 850 C; several months at
+40C
Mac Conkey agar – weak selective and differential capacities; protein
hydrolysate, lactose, bile salts (inhibits microorganisms excepting
Enterobacteriaceae), neutral red as pH indicator; lactose positives look red on this
medium

Media for anaerobes: reversed plate, with paraffin and oxygen reducing mixture
(pyrogalol, talc, potassium carbonate); Gas-Pack; candle jar;

Sabouraud medium – for fungi: glucose, protein digest, agar, pH 5, 6; preserved

in good condition for 2 months at + 4 0 C, in plastic bag; selective if antibiotics
added

Stuart medium – agar, Na thyoglycolate, Na glycerophosphat, Calcium chloride ,

Methylene blue; preserved in good condition for 2-3 months at + 4o C; universal
transport medium, survival of bacteria for 1-3 days
PW7. Culturing microorganisms

Instruments:
- Bacteriological loop
- Pasteur pipette
- Cotton swab mounted on a wood or metal stick
- Glass L stick
- Others

Heating the loop at red and flaming of the wood support

1. the pentagon method (the streak plate method) – the inoculating
loop is heated between the streaks to obtain single colonies
2. Inoculation with the calibrated loop: 0,001 ml
3. Successive diluting technique
4. Inoculation with the L glass stick
5. Inoculation with the cotton swab – e.g. for AST (Antimicrobial
Susceptibility Testing)
6. Inoculation with the simple, right loop, into the depth of the
medium
7. Inoculation with the Pasteur pipette
8. Inoculation by flooding
9. Successive dilutions technique (binary, decimal etc.)
 Special techniques:
1. Heating on water bath at 80 la 0 C of the sample inoculated in VF
broth, for isolating sporulate anaerobes
2. Adding Ethylic alcohol 95 0 C and shaking for 1 h at room
temperature, for isolating sporulate anaerobes
3. Biological methods – animal inoculation (e. g. Str. pneumoniae on
white mouse)

PAY ATTENTION:
We write on the plate: identification number, date, type of sample
Plates are dried bottom up in the incubator, half opened, before
inoculating them.