Characterising new bacterial colonies Flashcards
What are the 7 different methods?
Morphology = study of shape, size, structure
Physiology = conditions required for growth
Biochemistry = tests for specific enzymes/metabolic pathways
Serology = detecting antigens with antibodies
Bacteriophages
Pathogenicity
DNA analysis
What is colony morphology?
What do you look for?
What causes change in colony morphology?
What is cell morphology?
What are you looking for?
What are the 4 different shapes?
What is required?
Studying appearance of a colony under specific conditions in agar
Colour, shape, texture, size
1 gene
Appearance of cell under microscope
cell arrangement = dependent on the pattern that cells divide
cocci = spherical
rods
vibrio = comma shaped
spirillum = spiral
Microscope & fix bacterial samples
What is fixation? What is this an advantage for?
What is heat fixation? What are its pros & cons?
What is chemical fixation? What are its pros & cons?
What are the 4 steps briefly of preparing the slide?
What has to be placed on the slide when examining it with a x100 objective lens?
When spreading the culture over the slide, what must you ensure?
Kills & preserves samples on slide- pathogens
Heat slide gently.
+ preserves overall morphology
- doesn’t preserve delicate structures
incubate with formaldehyde fumes or ethanol which reacts with cellular components & makes them inactive & insoluble
+ preserves fine cell structures
- Prepare smear
- Heat/chemical fix
- Staining
- Microscopy
Drop of oil
Not too thin (can’t find bacteria) & not too thick (to see individual cells)
What to dyes do?
What are the 2 types of ionisable dyes and examples?
Acid-fast staining——-
What does it stain?
What are the 5 steps to staining with it?
What do the target bacteria look like? What do other bacteria look like?
Gram staining——-
What does it stain?
What are the 4 steps?
What do the gram positive & negative bacteria look like? Why?
Chromophore that binds to specific cell components
Basic: methylene blue, crystal violet, malachite green
Acid: rose bengal, carbol fuchsin
Acid-fast/mycobacteria with high lipid contents in cell walls
- Fix the smear & hot carbol fuchsin
- Wash with water
- Add 20% sulfuric acid for 10 minutes
- Wash with water
- Counterstain/destain with methylene blue
Target = red, rest = blue
Gram positive bacteria with lots peptidoglycan
- Heat fix smear & crystal violet
- Add iodine/KI (forms complex in cell walls)
- Wash with solvent 95% ethanol
- Counter stain with other colour e.g carbol fuschin
purple = gram +ve
pink/red = gram -ve
More peptidoglycan took up violet/darker stain
What different conditions are required for growth?
What nutritional requirements would you consider?
What about light?
Why is temperature important?
What are the difference between psychrophiles, mesophiles & thermophiles?
Nutrion, light, temperature, pH, oxygen, pressure, tolerance to chemicals
How does it get carbon/nitrogen (trophs), does it need vitamins/amino acids?
is it photosynthetic? some are sensitive to UV light
Enzymatic reactions- high rate with increased temp but can denature
Psychrophiles = like the cold- need more unsaturated fats in the membrane to be fluid at low temp
Mesophiles = intermediate temp
Thermophiles = like high temp
What are the following philes and where do they prefer?
halophile?
Acidophile?
Alkaliphile?
What kind of halophile is Halamonas titanicae?
Like saline conditions
Acidic/low pH conditions- volcanoic soils/springs & sulphur/metals
high pH- soda/alkaline lakes
iron-eating
Oxygen——
To what extent do the following types of organisms need oxygen?
Obligate aerobes
obligate anaerobes
facultative aerobes
aerotolerant anaerobes
microaerophiles
What has desulfovibrio vulgaris evolved to do?
What experiment can you do to distinguish these different organisms?
What is the oxic and anoxic zone?
need O2 for growth
O2 is toxic so don’t need it for growth
grow without O2 but use it if available
grow with O2 but don’t need it
grow at low O2 levels
doesn’t use O2 as an electron acceptor- uses reduced sulfate so breathes it instead
Grow them in test tubes with soft nutrient agar bottom layer and O2 in top layer
oxic zone = zone where O2 penetrates agar (at top of agar)
anoxic zone = zone where o2 does not penetrate agar (rest-bottom)
Presure——
what is the name of an organism that grows in high pressures & high temperatures?
where abouts do they inhabit?
what is the name of an organism that grows in high pressures & low temperatures?
What does pressure affect?
for every 10m depth in the sea, how much does the pressure increase?
why is it difficult to create these conditions in a lab?
barophiles which grow at high pressure & as a result temperature
thermal deep sea vents
psychrophile
enzyme-substrate binding, membranes, transport
1atm
expensive & dangerous
What kind of inhibitor is sodium azide?
what does it do to gram negative bacteria?
adding this to a mixture of gram+ve and -ve bacteria, what would you end up with?
what are chaotropic agents?
phenol extraction is used to extract DNA & RNA from samples, why can’t you do this with Pseudomonas?
how can you use antibiotics & its varying sensitivities in bacteria to characterise them?
respiratory
inhibits cytochrome c oxidase so blocks the active site
resistant gram +ve bacteria
cause denaturation of macromolecules
resistant to phenol as contain enzymes that break down benzene ring & use as food source
- score minimum inhibitory conc of antibiotics- wells of different species with decreasing antibiotic concentration x2- quantify & compare to data sets
- antibiotic-impregnated discs- measure radius & compare to data sets
Sugar metabolism tests—–
how do anaerobic bacteria metabolise glucose?
how do aerobic bacteria metabolise glucose?
what test can you use for determining the products of sugar fermentation?
what does a durham tube do?
what is the positive test?
release CO2, acetaldehyde -> ethanol
release CO2 & water
take nutrient broth with the culture medium & incubate with a Durham tube with a pH indicator
captures the gas produced
yellow = anaerobic (produced carbonic acid) pink/red = aerobic
What is another test for determining if a bacteria is aerobic or anaerobic?
What makes up the test?
What happens to the O2 gradient?
if you have aerobic bacterium, what happens?
anaerobic bacterium?
what is the colour change?
Oxidation/fermentation test
Tube with soft agar, gap at top of air, glucose & pH indicator
diffuses from top of tube = oxic zone
grow in oxic zone = yellow
ferment & grow throughout medium = whole medium is yellow
starts orange, areas with bacteria growing = yellow
What does the catalase test determine?
What is the procedure?
what is a positive test?
negative?
If bacterium contains catalase to break down H2O2
take H2O2 & add to cell suspension on glass slide
bacterium containing catalase = bubbles from breaking down H2O2 into O2 and H2O
no bubbles- doesn’t contain catalase
what does the urease test determine?
what is a positive test?
if the bacteria contains urease to convert urea into ammonia
if urease present, ammonia is produce which causes increase in pH & so colour change with indicator
Serology test with antibodies to detect antigens———
How can you generate antibodies for the antigen?
What happens when antibodies recognise antigens?
How do you perform the tests?
what is a positive & negative result?
how can you quantify this test?
Purify antigen, inject animal & collect & purify these antibodies made
agglutination
- in wells of microtitre plates, add purple stained bacteria
- add different antibodies into each well
positive = agglutination so bacteria clump together
negative = cells settle to bottom of well- not visible
add fluorescent labels to antibodies & use plate reader to measure level of agglutination
how can you use bacteriophages to characterise bacteria?
how can you use pathogenicity to characterise bacteria?
what different DNA analyses can you use to characterise bacteria?
what are the pros of each?
how can you overcome problems for bacteria that can’t be cultured?
bacteriophages have narrow host range- tend to only affect 1 species- plate bacteria as lawn on agar & put drops of different phages & look for reduced growth after incubation for a clear zone
pathogens cause specific symptoms & have definite host range
genome sequencing = complete sequencing of genome but doesn’t give answer for functions
PCR = use to amplify specific DNA/16s rRNA sequences & compare to data bases of known bacteria
- genome sequencing = better to use sequence portion of genome & compare to database
+ PCR = sensitive, quick, easy
metagenomics- mass sequence analysis of genome DNA without isolating them & use algorithms to build the whole genome
