TERM 2 PRACTICAL - based on some lectures too! Flashcards
(16 cards)
Why do we do a serial dilution?
we do it to obtain a proper number of micro-organisms to obtain an easily countable number of colonies
in other words = if there is too much bacteria to count then you can do a serial dilution to have a manageable number of colonies so you can count from this
and then estimate the number of bacteria present in the original sample
what is SPREAD plating, what equipment do we use
Use T- or hockey-stock spreader ensure dilutions are mixed well
What are the streak plating objectives
To obtain well-isolated (single) colonies
To obtain a pure culture of bacteria from a
mixed culture
To propagate bacteria (grow and multiply the bacteria)
How do we calculate CFU/mL
NUMBER OF COLONIES / VOLUME PLATED X DILUTION FACTOR
Sugar fermentation test what colour does each one turn (positive and negative)
POSITIVE TEST = goes from colourless to pink
this means acid production from fermentation (gas accumulation in the durham tube)
Negative test = remains colourless, no fermentation and no pH change)
Urease test
what does urease break down into and what does this mean in terms of pH
some bacteria produce the enzyme urease which helps break down urea to ammonia (becomes more alkaline as increase in pH)
POSITIVE = PINK/RED
NEGATIVE = YELLOW
What are the 2 examples of urease positive tests ! WE HAVE LOOKED AT THIS IN OUR LECTURES
Helicobacter pylori
CAUTI (CATHETER ACCOSIATED URINARY TRACT INFECTIONS)
Test 3 - 3) Oxidative and fermentative breakdown of carbohydrates.
Colours for fermentive and oxidative
If it is oxidative = required oxygen
so one tube is yellow
If it is fermentative = don’t need oxygen
so both tubes are yellow
4) Test for Cytochrome Oxidase Enzyme
What do we do and results!!
Add a drop of oxidase reagent to filter paper. Pick off a colony and rub it into the reagent. Wait for 1 minute.
If oxidase positive = purple; If negative = no colour change.
5) Catalase Test
what do we do and how do you know its catalase positive and catalase negative?
Add a small amount of culture to a sterile inoculating loop.
Add a single drop of hydrogen peroxide to the culture.
This should be done over a discard bucket of Trigene.
If the reaction is positive, bubbles will form as hydrogen peroxide is converted to water and oxygen.
6) Citrate Test
prepare streak plate onto simmons citrate agar
Negative = GREEN = no bacteria
citrate positive = BLUE
How do we prepare a heat fixed stain
1) prepare the smear and dry this in the air
2) then heat fix the slide (3 passes 1 second iN BLUE FLAME)
3) GRAM STAIN (another q on this in a different slide)
4) microscopy - if you need 100 x = use one drop of oil
ensure you can see cells at 40X 1st
Gram staining steps
1) crystal violet
2) iodine
3) alcohol
4) saffrin
Explain the gram positive and gram negative stain
Gram-positive organisms thick cell wall of peptidoglycan, so retain the crystal violet stain when washed with alcohol. When safranin is added, it is retained but obscured by crystal violet. Cells stainpurple.
Gram-negative organisms have an outerlipopolysaccharidelayer. When alcohol is added these lipids dissolve, exposing the relatively thin peptidoglycan membrane. Crystal violent/iodine complexes can exit which decolourises the cell. Therefore, when the red safranin counterstain is added, gram-negative cell stainred/pink
Not all antibiotics work against all bacteria.
WHAT IS IT?
Beta-lactams such as penicillin (ampicillin..) work by mimicking the D-Ala-D-Ala peptide bond which holds layers of peptidoglycan together.
Penicillin binds to the penicillin binding proteins (PBPs) active site → stopping peptidoglycan layers from binding.
Eventually bacterial cell loses structural integrity and lyses = cell death.
Gram + bacteria = thick peptidoglycan layer and no outer membrane = affected much more!
Antibiotic with the biggest diameter
IS THE MOST EFFECTIVE
