6.1-6.6 Microbial techniques, Bacteria as pathogens, Antibiotics etc Flashcards
what is a ‘culture’
microorganisms that have been provided with the nutrients, level of oxygen, pH and tempertaure they need to grow in large numbers so they can be observed and measured.
what is a ‘medium’
a mixture of substances that promotes and supports the growth and differentiation of microorganisms.
What is aseptic technique
involves only introducing desired bacteria into the medium under sterile conditions in order to prevent the unwanted growth of organims.
why is aseptic technique important?
- other microorganisms may harm your culture or compete with it
- there may have been contamination with a pathogenuc microorganism
- even if you think the bacteria are non-pathogenic, there may be a mutant strain present that is
describe aseptic culture technique
6 points
this kind of q can be very differnt depedning on the specific task- so these are general pointers
(eg q Describe how you would use aseptic techniques to transfer bacterial cells growing on an agar plate to a tube containing a sterile broth)
- disinfect surfaces
- work near Bunsen flame
- flame the top of any tubes (or bottles)
- flame the inocculating loop
- open the lid of the medium for as little time as possible and only open it to the very minium you need
- allow the inoculating loop to cool down before you touch microogranisms with it as they will die if its too hot
What are the three types of medium
- agar (solid)
- broth (liquid)
- selective medium
what is a selective medium
medium containing a very specific balance of nutrients-this means only very specific bacteria will grow in it and mutant strands won’t
pros and cons of using broth media
pros:
can provide oxic or anoxic conditions depending on the depth which helps identify microbes by optimum conditions
also can grow large volume of bacteria
cons:
can’t get pure discrete colony for study
Describe the growth curve of a microogranism in a closed culture
lag phase
the microorganisms are adapting to their new environment and reproduction rate increases slowly
log (exponential phase)
microorganisms grow at their maximum arte as long as there are sufficient nutrients
stationary phase
death rate=reproduction rate (due to build up of toxic waste products and waining nutrients)
death phase
death exceed new cell population as conditions continue to deteriorate
how can bacterial growth be measured (3 ways)
- cell count
- tubidimetry
- dilution plating
what things could you do to identify a specific bacteria
(- first you need to isolate it from the pateint (vomit/faeces/ food they consumed if it way say salmonella) )
- look at the colonies to see if they have a characteristic of said bacteria (like certain shapes texture colour etc)
- use gram stain to show presence of gram positive or negative bacteria (staph eg gram +ve)
- grow on selective media that identifies that bacteria (staph) or eliminates other bacteria
- use antibiotics against that bacteria
in a question it would likely tell you the type of bacteria, relate each type of test back to THAT BACTERIA its in CURLY BRACKETS so eg use antibiotics against Staphylococcus
be obvious - really spell it out
how does streak plating let you isolate bacteria
3/4 points
- culture/colonies is spread out on the agar plate
- because this seperates out individual bacteria
- so that colonies are discrete/seperate
- so only one type of bacteria can be picked up
how can cells be counted
using a haemocytometer
what is a haemocytometer
a thick microscope slide engraved with a grid and a rectangular chamber that holds a standard vol of 0.1mm3
what is a c-chip haemocytomter
a haemocytomometer that is
- disposable
- non-breakable
- reusable within limit
how can haemocytometers be used to count only viable cells?
trypan blue is used as a stain which stains dead cells blue
dilute your sample with trypan blue in 1:1 ratio
advantages and disadvantages of cell counting (with haemocytometer)
+ve: you can distinguish between viable and non-viable bacterial cells
-ve: time consuming and equipment is expensive
how do you count cells on a haemocytometer (what counts as in and which squares do you count)?
if a cell overlaps a line, count it as “in” if it overlaps the top or right-hand line and “out” if it overlaps the bottom or left-hand line
count in the 4 corner squares and the central big square
how does tubidimetry work?
it is a specialised form of colorimetry. As turdidity increases, transmission descreases and absorbance (measured in Au) increases.
This value can be linked to cell count by measuring absorbance of samples with a known cell count (via counting cells with a haemocytometer or using dilution plating) and using a calibration graph to obtain the cell count in an unknown sample.
define ‘turbid’
opaque/cloudy/thick with suspended matter
advantages and disadvantages of using turbidimetry to measure bacterial growth
+ve: quick and can be conducted in the field
can be used to measure large quantities of bacteria
-ve: equpiment expenisve
counts non viable cells
calibration curve required to obtain actual cell count
assumes denisty is same across culture (magnetic stirrer used to help this)
how does dilution plating work?
straight after culturing, colonies cannot be counted because a single mass is often present.
so that single colonie can be seen, the original culture is serially diluted, a spread plate is made and the colonies can be counted. This is then multiplied by the dilution factor to obtain cell count.
formula to caluclate dilution factor
diltuion factor = volume of sample added/ total volume
what is the principle that diltion plating works on
every colony is grown from a singke viable microorganism
advantages and disadvantages of dilultion plating
+ve: doesn;t require complex or expensive equipment
only counts viable cells and obtains a direct count
-ve: slow because an incubation period is needed (to allow for the deelopment of visible colonies) and serial dilutions required.
how does bacteria act as an agent of infection?
3 ways
- exotoxins
endotoxins
host tissue invasion
what is an exotoxin
+ eg
soluble proteins produced and released by bacteria as they metabolise and reproduce
eg: Staphylococcus
what are endotoxins?
+ eg
lipopolysaccharides in the outer lipid membrane of gram negative bacteria
these are only released when the bacteria is broken down or damaged or lysed
eg: salmonella/e.coli
how much does it take of an
- exotoxin
-endotoxin
to cause symptoms
exotoxin: requires very small quantities are required to cause symptoms (will happen fast)
endotoxin: requires being present in large amounts to cause symtpoms (happens slow because bacterium needs to be broken down first)
what is an example of a disease caused by host tissue invasion
what bacterium is it caused by
tuberculosis (TB)
caused by mycobacterium tuberculosis
how does TB infection work?
- infects phagocytes (macrophages) in the lungs
- first infection is symptomless.
- infected phagocytes are sealed in tubercles as a reuslt of an inflammatory response in the lungs
- bacteria lie dormant inside the tubercles
- they are not destroyed by the immune system as the tubercles are covered with a thick waxy coat
- when the immune system becomes weaked, the bacterua become active again and slowly destroy lung tissue
symptoms of TB
fever
fatigue
coughing
lung inflammation
if untreated will cuase extensive damage to the lungs which can result in death due to respitory failure, it can also spread to other parts of body
what are antibiotics
substances which can inibit the growth of or destroy bacteria
2 types of antibiotic:
bacteriacidal
bacteriostatic
bacteriacidal antibiotics
what are they
how do they work/ what do they work on
give example
bacteriacidal antibiotics kill bacteria by destroying their cell wall thus causing them to burst.
target cell wall so work best against gram positive organisms (narrow spectrum)
penicillin
bacteriostatic antibiotics
what are they
how do they work/ what do they work on
give example and what does that specific antibiotic do
they inhibit the growth and reproduction of bacteria
by stopping protein synthesis and production of nucleaic acids so the bacteria cannot grow and divide
this is broad spectrum as all bacteria do this stuff
eg: tetracycline (interfers with protein syntehsis in 70s ribosomes)
how is resistance to antibiotics controlled
- in hospitals new patients are screened on arrival, trated and isolated if infected to prevent spread
- antibitoics are only used when needed
-whole course of antibiotics completed - all stagg must follow strict hydgeine regimes (washing hands with alochol based antibacterial gels and wearing suitable clothing
why is it important that antibitoic courses are completed
to ensure that all the bacteria are destroyed including the more resistant strains
and to minimise selection pressure on baceria to prevent resistant strains from forming
influenza
- transmission
- mode of infection
- pathogenic effect
transmission: droplet infection, direct contact with virus-filled muscus, direct contanct with animal infection, direct contant with infected surfaces
mode of infection: infects ciliated epithelial cells of the lungs (antigen fits into receptor on host cells, injects viral RNA), viral RNA takes over cell biochemistry, cell produces new virus particles, cell lyses, many virus particles released. (lytic cycle)
pathogenic effect: headache, sore throat, sneezing, fever 5-7 days, vomiting, muscle pain
treatment: antiviral med, painkillers
puccinia graminis (stem rust fungus)
- transmission
- mode of infection
- pathogenic effect
transmission: wind carries spores from infected plats, infected fragments left in soil from the two hosts (cereal crops and berberis
mode of infection: spore germinates in water on plant, prouces hyphae which enter the plant through the stomata, hyphae grow into mycelium surround all tissues in the plant, produce enzymes like cellulase to digest plant and all the nutrients are absorbed into the fungus
pathogenic effect: nutirents lost to fungus, weakened stem, water loss as the plant can’t control transpiration (redued photosynthesis, pustules on epidermis which eventually burst to release more spores.
malaria
- transmission
- mode of infection
- pathogenic effect
transmission: transmitted through the vectore of the female Anopheles mosquito when she feeds to get protein to lay eggs
mode of infection: parasite transmitted via mosqutio, travels to liver infects rbcs reproduces asexually inside erythrocytes and causes lysis
pathogenic effect: malara bursts out of rbcs every 2-3 days causing, sweating, shaking, muslce pain, headaches liver damage and anemia
Why are antibiotics not effective against viruses or mammalian cells? (4)
- viruses do not perform metabollic processes for (bacteriostatic) antibitoics to target
- viruses do not have a peptidoglycan cell wall for (bacteriostatic) antibiotics to taget
- mammalian cells do not have a peptidoglycan cell wall for (bacteriostatic) antibiotics to taget
- mammalian cells have 80S ribosomes not 70S ribosomes for antibiotic cells to target
State two differences other than toxicity between endotoxins and exotoxins (3 points)
- endotoxins released from gram negative bacteria (only) but exotoxins released from both gram negative and gram positive bacteria
- endotoxins are lipopolysaccharides but exotoxins are proteins
- effect of endotoxins is later
