topic 6 Flashcards
[6.1]
Why must we use aseptic techniques when handling cultures of microorganisms?
To prevent:
● potentially harmful microorganisms escaping from your cultures into air + infecting humans
● microorganisms in the air entering and contaminating your cultures
aseptic techniques:
● all equipment (including agar + petri dishes) should be sterile
● flaming equipment in a bunsen flame ensures sterility
● inoculation should be done with a flamed instrument
● lids should be replaced as quickly as possible
● work near a bunsen burner flame
● disinfect surfaces
Culture?
when microorganisms are provided with the nutrients, level of O2, pH + temp they require to grow large numbers, so they can be observed + measured.
steps + techniques involved when culturing microorganisms:
- obtain culture from microorganism
- provide it with right nutrients + environment (eg carbon,nitrogen,protein,minerals) needed to grow
- transfer to the nutrient medium (nutrient broth [liquid] / nutrient agar [jelly] ) using sterile inoculating loop
- close dish airtight
inoculating when using a nutrient broth..
-make a suspension of the bacteria to be grown + mix a known volume of it with the sterile nutrient broth in the flask
- close quickly (cotton wool) to prevent contamination w air
- flask incubated at suitable temp + is often shaken regularly to allow O2 to the growing bacteria
inoculating when using nutrient agar….
if inoculating agar, either:
- Make a streak plate
- Make a spread plate
streak plate?
Sterilise inoculating loop by flaming, dip in culture, sterile plate, at least 3 streaks straight / zig-zag, turn, streak which must overlap with first streak, turn, streak to try to obtain single colonies.
: aim to obtain single colonies by rotating the plate to build layers of the culture on at least 3 separate streaks.
Spread plate?
distributing microorganisms evenly with a sterile spreader.
spread the sample evenly over the entire plate –> allow sample to absorb thoroughly (at least 5 minutes) before inverting the plate for incubation.
selective mediums?
medium containing a very specific balance of nutrients = only very specific microorganisms with those particular requirement will grow in it = mutant strains won’t
= SM important in identifying particular mutant strains of microorganisms + antibiotic resistance
+ / - of using broth medium over agar?
+ Broth can provide anoxic conditions as well as oxygen closer to the surface = can provide information about what kind of oxygen requirements the microbes have.
+ They can also grow a much larger volume of bacteria.
- However, u can’t get a single, discrete, pure colony from a broth to inoculate with/study.
Different methods of measuring growth of bacteria?
- cell counts
- dilution plating
- turbidity (mass + optical methods)
In Cell counts, bacteria + single-celled fungi can be counted directly using a haemocytometer.
What’s a haemocytometer?
bacteria + single-celled fungi can be counted directly using a haemocytometer
= a thick microscope slide engraved with a grid + a rectangular chamber which holds 0.1mm3 volume of liquid.
- cell count
How to conduct a cell count?
- Cells counted using a haemocytometer
- The sample of broth is diluted 1:1 with trypan blue , which stains dead cells blue = so u can identify + count only the living cells.
- cells viewed + counted using microscope.
count cells in each of the 4 sets of 16 squares + take a mean.
Haemocytometer has been pre-calibrated so number of bacteria cells in 1 set of 16 squares = number of cells x 10^4 per cm3 pr broth. - This is repeated at regular intervals throughout growth = shows changing cell numbers.
+ / - of using cell count
+ useful because it counts only viable (living) cells + is accurate.
- slow
- equipment involved is expensive
- large margin for human error
- Diluting plating
- Dilution plating works on the principle that every colony is grown from a single, viable microorganism .
- Immediately after culturing, colonies cannot be counted because a single mass is often present.
= So that single colonies can be seen, the original culture is serially diluted, a lawn plate made and the colonies counted.
-This is then multiplied by the dilution factor to obtain a cell count.
dilution plating is used to find the total viable cell count.
What’s the total viable cell count?
A measure of the number of cells that are alive in a given volume of a culture.
+ / - of using diluting plating
+ only counts viable / living cells
+ no complex / expensive equipment needed
- slow as incubation period needed + serial dilutions needed
What’s turbidimetry?
Turbid?
Turbidimetry:
- Is a specialised form of colorimetry
= measures the concentration of a substance by measuring the amount of light passing through it.
Turbid:
- Is opaque, or thick with suspended matter (bacteria cells)
- Optical methods
[Turbidity]
- As turbidity⬆= transmission⬇+absorbance⬆ (measured in Au).
- This value can be linked to cell count by measuring absorbance of samples with a known cell count (by counting cells with haemocytometer / dilution plating), + using a calibration graph to obtain the cell count in an unknown sample.
+ /- of using turbidity measurement:
+ Quick + can be conducted in the field
- expensive equipment
- counts both living + dead cells
- Requires calibration curve from known samples
- Assumes density of cells is equal across culture
How to assess growth when fungi is used instead of bacteria?
measure diameter of the patches of mycelium
= used to compare growth rates in different conditions + find the optimum
Generation time?
The time between bacterial divisions
Analysing the data:
What are the different phases of a bacteria growth curve?
- Lag phase
- Log Phase / Exponential phase
- Stationary phase
- Death phase / Decline phase
- Lag phase
- when bacteria are adapting to new environment = not yet reproducing at their maximum rate
- Log phase
- When rate of bacterial reproduction is close / or at it’s theoretical maximum
- After EVERY round of division, population size doubles (exponential growth).
- Stationary phase
- When total growth rate is zero
- As number of new cells formed by binary fission = number of cells dying
Reproduction rate = Death rate
;so population size stabilises at its maximum
- Death phase
- when reproduction has almost ceased + death rate of cells is increasing
In death phase why does exponential growth in bacteria culture not continue?
(why do bacteria die?)
- reduction in amount of nutrients available - level of nutrients available = not sufficient/enough to support more growth + reproduction
- Build up of waste products - inhibits further growth + could poison + kill culture.
- CO2 produced by respiring bacteria cells build up = pH of colony falls to point where bacteria can no longer grow.
[6.2]
Pathogens?
Types of pathogens?
microorganism that causes diseases + harm to other living organisms.
- Bacterial
- Viral
- Fungai
- Protozoa
Systemic infections?
Localised infections?
systemic: pathogen has spread through bloodstream = can affect many different organs = dangerous
Localised: pathogen is in 1 area only = less widespread effect
Why are bacteria described as agents of infections?
Bacteria can be agents of infections via production of
- endotoxins
- exotoxins
- invasion + destruction of host tissues / cells.
- Endotoxins:
Are lipopolysaccharides in outer membrane of gram negative bacteria cell wall
-The lipids (in lipopolysaccharide) act as the toxin
- The polysaccharide stimulates an immune response
- Endotoxins released from a bacterium if it breaks down (wall) + have effects local to the site of infection = effects show LATER (more time than exotoxins as bacteria cells needs to be destroyed first so they can leave)
Diseases caused by endotoxins are not fatal BUT symptoms they cause which can indirectly cause death. (eg dehydration due to severe diarrhoea)
Why don’t all antibiotics work with bacteria which release endotoxins?
Antibiotics which target cell wall, can cause more endotoxins to be released from gram negative bacteria, as the lipopolysaccharide component of cell wall is damaged + releases them.
= adverse effect
Case study of Endotoxin: Salmonella?
- bacteria invades lining of intestines + endotoxins cause inflammation
= the cells no longer absorb water = faeces become liquid + gut goes into spasms of peristalsis = results in diarrhea
How is salmonella spread?
- through ingestion of contaminated food + water
- salmonella bacteria live in guts of many food animals + can contaminate meat = if not cooked properly bacteria survive + pass into ur gut when u eat it
they survive stomach acid + pass into small intestines
Treatment / reduce transmission
- ensure meat cooked well
- washing hands after handling raw meat
- avoid contaminated water
- antibiotics reduces symptoms = feel better but act as a carrier for longer
- Exotoxins:
- soluble proteins produced + released from living bacteria as they metabolise + reproduce in cells of host
- produced by both gram +/- but mostly gram positive
- effects more widespread than endotoxins
- some damage cell membranes causing cell breakdown or internal bleeding
- cause most dangerous + fatal bacterial diseases
Case study of exotoxin: Staphylococcus?
- many have them in skin + gut flora = only cause disease if get inside tissues, if normal skin flora is changed or if person has compromised immune system due to other disease
- produce exotoxins which cause skin infections which can be fatal
- S. aureus: boils + styles (localised) + toxic shock syndrome (systemic)
- Host tissue invasion
bacteria can invade + damage cells of a host
Case study of host invasion: Mycobacterium tuberculosis
TB most commonly caused by Mycobacterium tuberculosis, spread by droplet infection.
TB often affects respiratory system, damaging + destroying lung tissue + suppresses immune system = body less able to fight disease
-⬆affects immuno-compromised individuals
symptom = coughing up blood + weakness
got 2 stages:
1. primary infection (no symptoms, just carrier)
2. active infection
The primary infection stage?
- Initial stage when M. Tuberculosis is inhaled into lungs, invades cells of lungs + multiplies slowly
- Immune system activated = localised inflammatory response - causes a tubercle (a mass containing dead bacteria + macrophages) = stays for 8 week in lungs
- often no symptoms in primary infection stage
