3.4 microbiology Flashcards
how are bacteria classified?
according to their shape, cell wall structure and their metabolic, antigenic and genetic features
what shapes are bacteria usually?
- spherical (coccus/cocci)
- rod shaped (bacillus/baccili)
- spiral (spirillum/spirilla)
how do they name bacteria?
- the name often reflects the disease they cause
- e.g pneumoniae which causes pneumonia
bacteria are further classified according to the structure of their cell wall. how is this determined?
using the Gram staining technique
how does the antibiotic penicillin work against bacteria?
- it prevents the cross-links from forming within the peptidoglycan layer
- and so weakens the cell wall in newly divided bacteria
are gram-positive or gram-negative bacteria most affected by penicillin?
- gram-positive
- penicillin prevents the cross-links from forming within the peptidoglycan layer, and so weakens the cell wall in newly divided bacteria
- so they are then subject to osmotic lysis, when water enters the bacterial cell causing the cell to burst
compare gram-positive bacteria with gram-negative bacteria:
GRAM-POSITIVE:
- thicker cell wall
- thick layer of peptidogkycan
- no lipopolysaccharide layer (LPS) so vulnerable to penicillin and lysozyme action
- peptidoglycan layer retains crystal violet stain so stains purple
- e.g Staphylococcus and Streptococcus
GRAM-NEGATIVE:
- thinner cell wall
- thin layer of peptidoglycan
- lipopolysaccharide layer (LPS) protects against penicillin and lysozyme action
- lipopolysaccharide layer prevents uptake of crystal violet stain, so only stains red once LPS removed and a counter-stain e.g safranin used
- e.g Salmonella and E.coli
do gram-negative bacteria contain peptidoglycan in their cell wall?
yes
- but due to the lipopolysaccharide layer, the crystal violet isnt retained
gram-Positive bacteria stain Purple and are susceptible to Penicillin due to the thick layer of Peptidoglycan
what is the Gram staining technique?
- transfer a small sample of bacteria to a glass microscope slide using an inoculating loop. pass the slide through a Bunsen flame a few times to fix the bacteria to the slide (it also kills them)
- add a few drops of crystal violet stain and leave for 30 seconds
- rinse excess using water
- add Gram’s iodine for 1 minute to fix stain
- bacteria which stain purple are gram-positive
to stain remaining bacteria: - wash with alcohol for 30 seconds to dissolve lipids in lipopolysaccharide layer and expose inner peptidoglycan layer
- re-stain using another stain e.g safranin, which stains unstained bacteria red
what does a growth medium such as nutrient agar have?
- nutrients - a source of carbon for respiration e.g glucose, nitrogen for synthesis of nucleotides and proteins, and vitamins and mineral salts
- water
- suitable temperature - 25-45°C for most bacteria; 37°C is optimum for mammalian pathogens. some can survive at 90°C (thermophiles) e.g Thermus aquaticus which evolved in hot springs
- suitable pH - optimum is slightly alkali (pH7.4) for most bacteria. some can survive acidic conditions e.g Helicobacter pylori in stomach (pH1-2)
- oxygen may or not be required depending upon the mode of respiration
what is it termed if a microbe needs oxygen for metabolism?
obligate aerobe
what is it termed if a microbe grows better in the presence of oxygen but can grow without it?
facultative anaerobes
what is it termed if a microbe cannot grow in the presence of oxygen?
obligate anaerobes
how do you ensure when culturing bacteria that only the desired bacteria is grown?
- by using aseptic technique
- so that you dont contaminate yourself or the environement
how is the equipment and media used to culture bacteria sterilised?
- heat at 121°C for 15minutes in an autoclave or pressure cooker, or by passing the equipment through a bunsen flame for 2-3 seconds until it glows red e.g an innoculating loop. this works for inanimate objects
- irradiation works well for heat-labile plastics
- benches cannot be sterilised but can be disinfected e.g with 3% Lysol, which reduces numbers of microbes, but not fungal spores
- living tissues cannot be safely sterilised without killing them so antiseptics are used to kill or inhibit microbes on the outside of living tissues only
why can’t heat-labile plastics be sterilised?
- they melt at the temperatures needed to sterilise them
- so radiation has to be used instead
pathogen definition
a disease causing microorganism
- sterilising kills all microorganisms including spores
- disinfection reduces the number of microbes
why is it important to grow bacteria at 25°C rather than 37°C?
- so that pathogenic microorganisms arent grown
(aseptic technique —> petri dish lids should be secured with tape but not completely so oxygen can still get in. all material should be safely disposed of afterwards by sterilising in an autoclave)
colony forming unit (CFU) :
- we assume that each colony grew from a single bacterium
what are the stages of a graph measuring bacterial growth?
- lag phase
- log phase (growth phase)
- stationary phase
- death phase
explain the lag phase of bacterial growth
population number increases very slowly because time is needed for enzyme synthesis
explain the log/exponential/growth phase of bacteria growth
- there are plenty of nutrients and few toxic by-products so there are no limiting factors
- this allows rapid reproduction
explain the stationary phase of bacterial growth
- cells are reproducing but population is relatively constant fluctuating around the carrying capacity, due to cell production equalling cell death
- the population has reached its carrying capacity because reduced resources (e.g nutrients/space/toxic waste products) are now limiting factors
explain the death phase of bacterial growth
- more cells are dying than are being produced so the population decreases
- death of cells is due to lack of nutrients, lack of oxygen or increased toxicity of the medium
what are the 2 ways in which bacterial growth can be measured?
- directly where the total number of cells is calculated
- indirectly by measuring the turbidity (cloudiness) of a culture
how can direct counts be counted when looking at bacterial growth?
- they can either be ‘viable counts’ where only living cells are counted or ‘total counts’ where both living and dead cells are counted, by using a haemoctyometer, originally developed to count blood cells
what does viable cell counts include?
- the number of living cells
when is viable cell counts particularly useful?
- in medical and food hygiene applications
why must a serial dilution be performed when working out the total viable cell count?
- as even in small cultures, the total viable cell count can exceed several million per cm^3
how is a serial dilution performed?
how is the viable cell count then calculated?
- is often done in tenfold steps
- i.e 1 in 10, but higher dilutions can be performed e.g 1 in 100
- for 1 in 10 dilution, 1cm^3 is added to 9cm^3 of sterile medium and mixed
- and repeated until a range of dilutions is obtained
- plate out each dilution
- incubate the plates at 25°C for 24-48hours to allow bacteria to grow
- the plages are examined and a plate chosen to count: the best one is a plate containing between 20 and 100 colonies (any more is difficult to count, and less than 10 has an increased error due to the small numbers involved)
- the viable cell count is calculated by multiplying the dilution factor by the number of colonies
- this technique has to assume that each colony originated from a single bacterium that divided asexually
- for dilution plate calculations:
- a common mistake occurs when 0.1cm^3 of sample is spread
- this represents a further tenfold dilution and so you should multiply the number of colonies by the dilution factor and then by 10
what are bacterial cell walls made up of?
peptidoglycan (murein)
define gram-positive bacteria
- bacteria that have a thick peptidoglycan wall and a purple appearance following gram staining
why do gram positive bacteria appear purple following gram staining?
- the thick peptidoglycan wall retains crystal violet
define gram-negative bacteria
- bacteria that have a thin peptidoglycan wall and an outer lipopolysaccharide membrane and a red appearance following gram staining
why do gram-negative bacteria appear red following gram staining?
- on treatment with alcohol, the lipopolysaccharide layer is lost and the crystal violet washes away
- the counter stain safranin stains the thin peptidoglycan layer red
what is an obligate aerobe?
- microorganisms/bacteria that (grow/divide/metabolise) in the presence of oxygen
what is an obligate anaerobe?
- an organism that can only survive in environments which lack oxygen
define facultative anaerobe
- an organism that GROWS best with oxygen
- but is capable of growing with or without OXYGEN
what are aseptic techniques?
- a range of techniques used to culture microorganisms under sterile conditions in order to minimise contamination
list the basic aseptic techniques:
- wipe surfaces with antibacterial cleaner
- set up bunsen burner nearby - convection current prevents microbes from entering culture
- flame inoculating loop and neck of bottles before use
- minimise time that vessels containing bacteria are open
- sterilise all equipment e.g use of an autoclave
- wear protective clothing
explain the difference between a spread plate and a streak plate
- spread plate - microorganisms distributed evenly with a sterile spreader
- streak plate - aims to obtain single colonies by rotating the plate to build layers of the culture on at least three separate streaks
what is a nutrient media?
- a solid or liquid nutrient-rich medium used in the cultivation of microorganisms
- contains a carbon source, nitrogen source, water and growth factors (e.g salts and vitamins)
describe how a viable cell count is conducted
- add a known volume of organisms to an agar plate
- incubate the plate
- count the number of colonies
what is assumed when conducting a viable cell count?
it is assumed that one cell gives rise to a single colony
what is the problem with the ‘one cell one colony’ assumption?
- it does not count for clumping of cells in the original inoculum
- this may result in a lower estimate of the number of cells
what is a serial dilution?
- a sequence of dilutions
- in which the dilution factor is constant, used to dilute a stock solution
suggest why when the microbiologist diluted the original sample by a factor of 10^-2, she could not calculate the number of bacteria per cm^3 [1]
- there were too many COLONIES to count / COLONIES (merged/clumping)
suggest why when the microbiologist diluted the original sample by 10^-6 a lower number of bacteria per cm^3 was calculated
- the extra dilution gives additional error / may not have mixed fully / inaccurate representation of whole sample / not valid to count less than 30 colonies / too few to be statistically significant
four agar types have resulted in different colony numbers because they contain different nutrients. state four ways that the agar types could differ in composition [2]
- different pH
- different C (source/concentration)
- different N (source/concentration)
- different growth factors
- different/different concentration vitamins/minerals
the type of bacterium that lacks lipopolysaccharides in its cell walls is a _____ bacteria
Gram positive
a corkscrew or helical shaped bacterium is known as a _____
spirillum
suggest why the bacteria labelled C in the diagram (a Gram negative bacteria) might be the possible cause of the food poisoning [1]
- (lipoprotein/lipopolysaccharide layer)
- protects against (some) (antibiotics) / penicillin / antibodies
- makes them less susceptible to attack by lysozyme
suggest why the number of bacterial colonies counted from an agar plate is likely to be an underestimate of the actual number of bacteria present? [1]
- doesnt include (dead/non-viable bacteria)
- cannot be sure that (each colony has grown from a single bacterium / colonies are not clumped)
suggest why these specific bacteria were cultured at 35°C and not 25°C? [1]
- need to count pathogenic bacteria
- pathogenic bacteria more likely to grow at room temperature close to body temperature
- want bacteria to grow quickly to identify to treat infection as quickly as possible
state one limitation of using a viable count method to monitor population growth of bacteria? [1]
- underestimate / doesnt allow for clumping
in both viable count and total count, name the procedure that the original culture requires in order to provide a final number within a countable range [1]
serial dilution
