lecture 4 Flashcards
what is the asceptic technique?
Reminder – aseptic technique used to ensure that cultures are inoculated only with microorganisms we want to study, keeping contaminants out
Aseptic technique is a set of procedures used when transferring organisms from one media to another – e.g. from an agar plate to a broth, from broth to broth, etc
Important first step is to flame the loop accurately to ensure sterility before organism transfer
Loop made of nichrome wire, heated in bunsen flame to kill any microorganisms before and after each use
work near the flame
Cultures must be sterile before the addition of the microorganisms of interest
Boiling media will kill many microorganisms but not endospores (see lecture 3)
Autoclaving generally performed to sterilise culture media (121°C, pressurised steam)
Heat-sensitive materials, e.g. blood, vitamins, antibiotics, are added to cooled autoclaved media – these can be made sterile by filtering
what is an organic compound?
microorganisms need organic compounds to grow. organic compounds are like methane. 1 carbon 1 hydrogen.
you would think that organic is carbon to carbon but urea doesnt have carbon in it. its just n2 h2 o2 bonded. not carbon.
how to microorganisms grow?
Cells undergo chemical reactions in a process called metabolism
These reactions are either energy-releasing (catabolism) or energy-requiring (anabolism)
Many microorganisms require organic compounds for energy – chemoorganotrophs
Most bacteria studied in microbiology are chemoorganotrophs, must be grown in media containing specific chemicals
Cells consist mostly of macromolecules and water
Dry weight of a prokaryotic cell typically consists of 55% protein (if a bacteria is made up of protein, we give them amino acids), 5% polysaccharide (sugars, maybe b vitamins to make its fats), 9.1% lipid, 3.4% lipopolysaccharide, 3.1% DNA and 20.5% RNA (nucleotides atcg’s, look at how its 20% because every 20 min they are busy multiplying).
These macromolecules made up of monomers, e.g. amino acids in the case of proteins, sugars in the case of polysaccharides, nucleotides in the case of DNA and RNA
Microorganisms require these monomers (nutrients) for growth and replication
These nutrients must be supplied in media used to grow microorganisms
what is peptone?
peptone is your source of proteins, your peptides, your small protein units with its amino acids. the yeast extract is a great source of carbs and the nucleotides.
Peptones are oligopeptides – short chains of amino acids, can be used by bacteria as both a nitrogen and carbon source
what is nutrient media?
All cells need a source of carbon for energy – most prokaryotes require an organic carbon source
Carbon often supplied in culture media using glucose, or using more complex mixtures e.g. peptone, yeast extract
The next most abundant element in the cell is nitrogen – used to produce proteins and nucleic acids
Nitrogen often supplied in culture media using amino acids, or more complex mixtures such as peptone
Peptones are oligopeptides – short chains of amino acids, can be used by bacteria as both a nitrogen and carbon source
what are the types of media?
Nutrient broth (NB) and nutrient agar (NA) often used as culture media – contains peptone, yeast extract and NaCl, pH adjusted to 7.2
This is an example of undefined/complex media – exact composition of media unknown, many bacteria can grow
Also known as general purpose or basal media
Chemically defined/synthetic media made by adding precise amounts of purified chemicals to water, the exact chemical composition is known
One example is minimal media, contains glucose as carbon source, ammonium phosphate as nitrogen source, and trace elements
how does one source peptone?
england use to source the best agar but when the mad cow disease outbreak they didnt make it anymore
how to we separate bacteria in mixed cultures?
Bacteria in nature rarely exist alone
When growing bacteria from samples, e.g. water samples, medical samples, etc usually a mix of organisms obtained
These organisms in the sample must be separated to study their individual properties
Solid media used to separate mixed cultures – as cells divide on solid media they pile up in one area, forming a colony
what is agar? around what temperature do you expect agar to set?
Koch first used potato slices to culture bacteria or solid media
Bacteria grew as colonies, each colony had a distinctive shape and colour, had grown from a single cell
Encountered problems – only some bacteria would grow on potato, frequent fungal contamination
Experimented with other types of solid media
Gelatin first used to solidify nutrient fluids, but wouldn’t remain solid at 37°C – optimal growth temp. for most human pathogens
Walter’s wife Fannie suggested using agar instead of gelatin. The fact that agar remains liquid to 45 degrees is also useful as heat-senstive chemicals, such as antibiotics, can be added to liquid agar before pouring
Agar is a polysaccharide found in red algae, was widely used in 1800’s as a setting agent
Agar superior to gelatin because:
it remains solid at 37°C
after boiling it remains liquid until cooled to about 45°C, allowing easy handling when making plates
What temperature do you warm blood to before adding aseptically?
blood is added asceptically, it has to be warmed to 45 degrees. if you add cold blood it will cause lumps to form/
what are the properties of agar?
Forms a strong gel when used as a 1% solution so its not expensive
Dissolves only in boiling water, once gel sets at around 45°C will only melt again at ~98°C
Agar can be incubated at up to 65°C without losing gel strength
what is the advantages of plastic petri dishes?
plastic dishes sterilised by irradiation or gassing
When agar is poured into the smaller of the two dishes, the larger one acts as a lid to prevent contamination. Petri dishes must be incubated lid side down and lids not labelled
The structure of the petri dish allows bacterial colonies to be exposed to air
what are the common types of solid agar?
Solid media is often complex, common types include: nutrient agar (NA) – same recipe as nutrient broth + agar
horse blood agar (HBA) – nutrient agar with blood added, some organisms require nutrients found in blood
‘chocolate’ agar (CHA) – nutrient agar with heated blood added (heated horse blood)
what are other types of media?
Enriched media – basal media with extra nutrients added, e.g. horse blood agar
if you wanna enrich media you add horse blood
Enrichment media – used to assist the growth of target organism in a mixed culture
contains additives which inhibit the growth of non-target organisms and improve the growth of target organisms
Differential/indicator media – contains substances which produce visible difference between organisms with different metabolisms
Used to visually identify target organisms growing in a mixed culture on solid media
Selective media: similar to enrichment media
enrichment media usually liquid, selective media usually solid
Selective media will restrict the growth of non-target organisms, may improve the growth of target orgs
MacConkey agar (MCA) is a selective media as well as a differential media Contains bile salts, which inhibit the growth of many microorganisms
The neutral red indicator in MCA differentiates between lactose fermenters/nonfermenters – red when acidic, yellow when basic
what type of microorganism is the primary cause of tinea? bacteria fungus virus amoeba
fungus
which of the following is not a eukaryotic cell? yeast amoeba virus algae
virus
the ability to see two objects as being separate is known as: refraction resolution magnification multiplication
resolution
a major constituent of gram-positive cell walls is: lipopolysaccharides lipoproteins penicillin peptidoglycan
peptidoglycan
flagella attached at many sites around the surface of a prokaryotic cell are called: monotrichous polar lophotrichous peritrichous
see how the clue was AROUND meaning peri. polar means at the ends. mono means just one. and lopo she wont used in an mcq test
mycobacterium can be best described as: acid-fast bacilli lacking a proper cell wall gram-positive bacilli gram-negative bacteria
mycobacterium cant stain (tuberculosis) so we have to use the afb stain
a suitable carbon source for culturing microbes is: an amino acid peptone carbon dioxide lactose
peptone
bacteria dont breathe carbon dioxide
an amino acid is for protein
lactose could be but isnt the best answer
macconkey agar acts as differential medium because:
fermentors look different to non-fermentors
haemolutic colonies look different to non-haemolytic colonies
salt requiring organisms grow
some bacteria will produce pigment on this agar
mackonkey agar has lactose. f the bacteria can ferment the lactose they turn pink making them look different to salmonella who cant ferment the lactose. mackonkey is not blood containing. it is clear (orangey clear) so it isnt haemolytic. does salt make it look different? no. colour does. when an agar is selective, it allows some organisms to grow and not others.
its not pigment that causes the colour, its fermentation that causes acid and its the pH indicator makes the colour.
bacteria that cause spoilage of refrigerated foods are called: thermophiles mesophiles psychrotrophs halophiles
psychrotrophs
which of the following bacteria cannot grow anaerobically?
staphylococcus aureus
escherichia coli
streptococcus pyogenes
pseudomonas aeruginosa
pseudomonas aeruginosa (that green slime organism) doesnt grow anaerobically
