Selective and Diagnostic Culturing L17 Flashcards
what is a phototroph
organism that is nutritionally able to synthesise all its required growth factors from simple substances
how do substances vary
depending on the nutritional classification of the organism
what are physical conditions that should be met
Temperature
pH
O2 levels
Osmolarity
what are general nutritional requirements
carbon source nitrogen source sodium phosphorus sulphur iron trace elements
what are the carbon sources available for nutrition
CO2 (AUTOTROPHS)
Organic molecules (HETEROTROPHS)
e.g. glucose
what are the nitrogen sources available for nutrition
inorganic e.g. N2 NH4
organic e.g. amino acids
what is sodium for in nutritional requirement
transporters
what are the phosphorus elements in nutritional requirements
nucleotides and ATP
what are the sulphur elements in nutritional requirements
SOv4^2- sulfate
H2S hydrogen sulphide
as aminoacids (Cys)
what are the iron elements in nutritional requirements
(Fe2+): cytochromes, catalase & other enzymes
what are the trace elements in nutritional requirements
Zn2+; Cu2+; Mn2+; Mo6+; Co2+
what are the trace elements for in nutritional requirements
required for a range of enzyme activities
required at very low levels (mg l-1)
sufficient present as contaminants in most media
what is defined media
made from mixtures of pure chemicals and contain precisely controlled amounts of each chemical
requires a thorough knowledge of cell metabolism
what is the growth media prepared as
mixtures of extracts:
- yeast extracts: vitamins and amino acids
- peptones: amino acids and TE’s from hydrolyzed protein
- meat infusions: soups
where are peptones produced
Peptones are produced from a variety of raw materials
what is the effect of a different growth media
end product will contain a different range of aa’s and minerals which favour growth of different organisms
what peptones are from milk
casein peptones
what peptones are from meat
general peptones
what peptones are from soya
plant peptones
what are the methods of isolating a particular organism
using
- selective medis
- differential medium
what is selective media
contains selective agents which prevent growth of some organisms but allows others to grow
what does selective media often contain
antibiotics
what does selective media require
requires an understanding of the growth parameters of the whole population
what is differential medium
particular organism has a characteristic appearance on the medium
which agar is used for staphylococcus aureus
baird parker
what does staphylococcus look like on baird parker agar, under what particular conditions
produces grey/black shiny colonies with a clear halo in 24h at 37°C
what do other staphylococci look like on baird parker agar
dont produce a clearing
what does bacillus look like on baird parker agar
brown, matt colonies
what does proteus look like on baird parker agar
produces brown/black colonies
what agar is E. coli and Salmonella grown on
MacConkey agar
how are E.coli and Salmonella differentiated on agar
pH change (media contains a pH indicator) due to their different ability to ferment lactose
what colour does lactose positive turn and which bacteria on what agar
pink
E.coli
MacConkey
what colour is lactose negative
yellow
what is bacillus cereus selective agar used for
Precipitates Lecithin in egg yolk
Cannot utilise mannitol so medium stays blue
what bacteria grows well on mannitol salt agar, what does the bacteria do
Staphylococcus aureus grows in presence of high salt (S) and ferments mannitol to produce acid (D)
agar changes from normal red to yellow
what are the three general modes of antibiotic action
1) prevent synthesis of bacterial cell wall (e.g. all β-lactams)
2) damage cytoplasmic membrane (e.g. polymxyin)
3) interfere with biochemical synthesis
what does Rifampicin do, where is it used
inhibit transcription
antibiotics - interfere with biochemical syntheis
what does Chloramphenicol, Streptomycin, Tetracycline do, where is it used
inhibit ribosome function
antibiotics - interfere with biochemical synthesis
what does Fluroquinolones do, where is it used
inhibit DNA replication
antibiotics - interfere with biochemical synthesis
what can lead to antibiotic resistance
1) Absence of a target site
2) Variants/point mutations in target sites that prevents the antibiotic binding
3) Enzymes to detoxify the antibiotics
4) Efflux pumps that remove the antibiotic before it damages the cell
when are enrichment procedures used
Used where essential to detect very low numbers of a specific organism
e.g. food borne pathogens in food samples
Also helps to revive damaged cells
e.g. those that have survived a processing step or have been in an unfavourable environment and may be sub-lethally injured
what is the enrichment procedure steps STEP1
Pre-enrichment
increase cells present
allows resuscitation/recovery of damaged cells
non-selective broth used
what is the enrichment procedure steps STEP2
Selective enrichment (broth)
promotes desired organism growth
prevents/retards growth of other organisms
combinations of:
substances toxic to unwanted bacteria
inhibitory growth conditions
conditions increasing growth of target organism
what is the enrichment procedure steps STEP3
Selective plating (agar) isolates organism of interest on selective /diagnostic agar = presumptive positive
what is the enrichment procedure steps STEP4
Confirmative tests
further testing is required to confirm identification
what are confirmatory tests
1st 2 common tests are Catalase and Oxidase tests
what does the catalase test do
detects enzyme that detoxifies Hydrogen peroxide
Typical of obligate aerobes and most facultative anaerobes
what does the oxidase test do
detects cytochrome c oxidase
Tells you that organism is able to use oxygen as a terminal electron acceptor
what is defined medium
ability to utilise specific sugars or amino acids
what is medium containing defined substrates
ability to metabolise specific compounds
what is the more common small scale rapid tests
API or Biolog
what biochemical tests are carried out by preparing small cultures
defined medium
medium containing defined substrates
more common to carry out small scale, rapid tests e.g. API or Biolog
what is antibiotic testing
serology
what happens in antibiotic testing, serology
Agglutination tests carried out using antisera specific for the target organism
what does it mean if clumping occurs in serology
cells have cross-reacted with the antibody
what DNA based tests detect specific genes
DNA hybridisation methods
PCR amplification of genes
how is media prepared
Components of media provided as dry powders
- Correct amounts weighed out
- Mixed with correct amount of distilled water
- Sterilised by autoclaving
- Cool and pour into Petri dishes
> Before pouring, heat sensitive components can be mixed into the molten agar
> Includes many antibiotics, vitamins, proteins, lipids
