Microbiology Flashcards
What is the purpose of a bacterial cell wall? What is it made of and what can they be classified as?
-prevent osmotic lysis
- give rigidity and cell structure
- made of peptidoglycan
- classified as:
GRAM POSITIVE
GRAM NEGATIVE
What is gram positive and why does it happen?
- cell wall stains purple due to take up of crystal violet dye
- thick peptidoglycan with one inner peptidoglycan bilayer
- ethanol does not remove
- more susceptible to antibiotics etc
What is gram negative and why does this happen?
- cell wall does not stain purple as ethanol washes out purple dye
- turns red/ pink due to counter-dye ( safranin)
- inner= peptidoglycan bilayer, middle= thin peptidoglycan, outer= lipopolysaccharide
- protects from lysosome, antibiotics
What is the explanation for the gram stain?
- crystal violet stains peptidoglycan
- iodine forms crystal violet- iodine complex in wall so it cannot be washed out
- ethanol removes dye from gram negative, gram positive retains dye
- gram negative stained with counter-dye- safranin to be seen under microscope
How do pour plates with antibiotics work?
- antibiotic disk (penicillin) placed in middle of pour plate
- antibiotic diffuses from disk through agar and kills bacterial colonies it contacts
- clear ring around disk where bacteria killed
- kills best= larger diameter
How is bacteria grown on a streak plate?
Isolate pure colonies by mechanical/ physical means
inoculating loop, streak- dilute- streak- dilute etc until very diltue
What are the physical metabolic requirements for bacteria?
- optimum pH
- oxygen availability
- optimum temp
- pathogens (mesophiles) = body temp
- saprotrophs ( feed on dead matter) = lower temps
- psychrophiles= lower temps ( down to 15 degrees)
- thermophiles= higher temps (up to 80 degrees)
What are the nutrient requirements for bacteria?
- carbon source (eg glucose—> respiration)
- nitrogen source (inorganic- nitrates, organic- amino acids)
- inorganic ions (phosphate ions—> ATP, DNA)
- vitamins (growth factors)
What are the three types of oxygen requirements for microbes?
OBLIGATE ANAEROBES - only grow in absence of oxygen OBLIGATE AEROBES - only grow in presence of oxygen FACULATIVE ANAEROBES - grow best with oxygen but can grow without
What are the two types of growth media for microbes and what is needed in them?
AGAR (SOLID) BROTH (LIQUID) - both have C source (glucose) - both have N source (amino acids) - also have specific vitamins and minerals
Why do we count bacteria?
1) water supply (uncontaminated)
2) environmental health monitoring ( sea water at beaches)
3) monitoring growth in fermenters (checking for unwanted bacteria)
What are three problems and their solutions for counting bacteria?
1) small- use microscope
2) lots of them- dilute
3) alive or dead?- allow to grow in colonies as only living ones can
What are the two types of direct counting and what are the pros and cons of each?
1) viable cell count- JUST LIVING- dilute
SERIAL DILUTION
+ counts living cells
- possible to underestimate pop (dilute too much)
- takes a long time for bacteria to grow
- if mixed with other culture that takes longer to appear then won’t be present when counting
2) total cell count- LIVING AND DEAD- haemocytometer
HAEMOCYTOMETER
(grid under microscope)
+ quick
+ counts several types of cell
- difficult to count in groups
- need oil immersion lens
- counts living and dead
What is the one type of indirect counting, how is it done and what are the pros and cons?
1) total cell count- turbidimetry (cloudiness)
METHOD- measure cloudiness and estimate no of bacteria
- calorimeter measures light absorbed
- more light absorbed= higher turbidity= more bacteria
- compared to standard cells
TURBITIMETRY
+ quick
+ counts several types of cells
- counts living and dead
-doesn’t discriminate between non-living matter and bacteria
How do you work out dilution factor?
no. of mircrobes counted on plate x dilution factor= bacteria in original sample of 1cm
How is the serial dilution method carried out?
1) 9cm3 of sterile H2O in 6 sterile test tubes
2) sterile pipette, add 1cm3 of culture to first tube
3) sterile pipette, transfer 1cm3 from one tube to another
4) repeat for all tubes, mix thoroughly
5) use glass spreader, spread all over plate
6) repeat for all dilutions and then seal plates
7) incubate at 25 degrees for 24 hours (colonies to grow)
8) choose dilution with reasonable no. of colonies
9) count colonies for three plates and calculate mean
10) mean x dilution factor= original sample
Culture is handled in a way that prevents:
- contamination of culture
- contamination of people
- contamination of envrionment
How are plastics and glassware sterilised?
PLASTICS irradiated GLASSWARE autoclave (pressure cooker) pressure of 15 atm, temps 121 degrees 15 minutes to kill bacteria and spores
what sterile precautions need to be taken?
- don’t seal petri dish- allows in O2- anaerobic pathogens not wanted
- dont incubate at 37 degrees- encourages human pathogens
6 sterilisation techniques and their reasons
1) autoclave- kill bacteria and spores
2) flame inoculating loops and glass spreaders- kill bacteria
3) open petri dish at small angle- prevent bacteria entering
4) roaring blue flame- convection currents lift air away from culture
5) don’t put caps on desk- contaminate desk
6) flame bottle neck- convection lifts air from broth
What are the shapes and groupings of bacteria called?
cuboid= bacillus sphere= coccus wiggly= spirillum
sheets/ clumps= straphylo
pair= diplo
attached in chains= strepto
squares= tetrads
