Microbiology🔬 Flashcards
What are the different bacteria shapes?
- Cocci - spherical
- Bacilli - rod
- Spirilla - spiral
- Vibrios - comma
What are archaebacteria?
Bacteria which thrive in extreme environments
What are eubacteria?
Found in all BUT extreme environments
What is gram staining?
LOOK AT DIAGRAMS
- Heat fixed smears of bacteria are stained with crystal violet solution and then washed in ethanol
- Decolourises gram-negative bacteria
- Smears are stained with safranin (counterstain)
- Stains gram-negative bacteria pink/red
- Stains gram-positive purple (retains crystal violet)
How do bacteria multiply?
•Binary fission - asexual reproduction
•Keep doubling each generation
- exponential growth
What do bacteria need to have when grown?
•Suitable physical conditions -temperature (usually >_ 25’C) -suitable pH (usually 7.4) •Water •Nutrients -carbon, glucose, nitrogen, amino acids, inorganic ions, vitamins
What are obligate aerobes?
Require oxygen for metabolism/growth
What are obligate anaerobes?
Can only survive in the absence of oxygen
-oxygen inhibits growth and metabolism
What are facultative anaerobes?
- Grow better in presence of oxygen but can slowly grow without
- Make ATP by anaerobic respiration of oxygen is present - but able to switch to fermentation or anaerobic respiration if oxygen is absent
What happens in the lag/latent phase?(1)(Population growth curve for bacteria grown in a culture)
- Cells are active but bacteria are unable to divide
- Cells take in water and synthesise ribosomes and enzymes
- Length of phase depends on medium used and whether cells were growing in a similar medium before
What happens in the exponential/log phase?(2)(Population growth curve for bacteria grown in a culture)
- Nutrients are plentiful
- Cells are very active and reproduce at fastest rate
- When cells are multiplying at max rate they are ‘in a state of balanced growth’
What happens in the stationary phase?(3)(Population growth curve for bacteria grown in a culture)
- Cells alter the culture medium as they grow
- Nutrients become depleted and there is a fall in pH - build up of CO2, acids and other metabolites
- Reproductive rate falls and cells did in greater numbers
- Limiting factor
What happens in death phase?(4)(Population growth curve for bacteria grown in a culture)
•More cells die than are produced •Number of living cells declines -starvation -shortage of oxygen -toxicity of waste products
What is a haemocytometer? (Total count)
- Special slide developed for blood cell counts
* Able to count bacterial cells in known volume of liquid and therefore calculate concentration ->no. per cm3
What is turbidity? (Total count)
- Cloudiness of culture
* Degree of cloudiness as measured with colorimeter proportional to concentration
What is the assumption of serial dilution? (Viable count)
•That one colony is produced from one bacteria
•This may underestimate the number of bacteria
-clumping if cells causes colonies to merge
How do you ensure that only the microorganism under investigation is cultured?
•Aseptic technique
-involves handling cultures in a way to prevent contamination by unwanted organisms
•Also prevents contamination of personnel and the immediate environment
Key points - Aseptic technique
- Hands and surface cleaned before and after
- Sterile agar and Petri dish
- Work by a bunsen burner to give updraft->helps prevent airborne particles landing on agar
- Mouth of culture bottle is flamed to kill unwanted microbes
- Lift petri dish lid at shallow angle to protect agar surface
- Flame sterilise inoculating loop/use sterile equipment
- Autoclave plates/equip to sterilise at end of work
What is asepsis?
State of being free from disease-causing microorganisms
What is sterilisation?
Removal or destruction of all living organisms, including spores
How are bacteria grown?
- Cultured in a liquid broth or on solid agar
- Medium is sterilised and contains specific nutrients
- Inoculation - sterile loop is dipped into bacteria source and then into culture medium
What is total count?
- Includes both living and dead cells
- Can be measured by haemocytometry
- Numbers can be overestimated in a population due to inclusion of dead cells
What is viable count?
- Includes only living cells
- Involves growing bacteria to form distinct colonies that can be counted
- Numbers can be underestimated because of clumping of cell’s when the plates are made - serval cell’s forming a single colony
SEE DIAGRAMS FOR SERIAL DILUTION
SEE NOTES
Both gram positive and gram negative bacteria stain purple. Why do gram negative bacteria not appear purple?
Their cell walls do not retain the crystal violet stain.
Instead, they are stained red by the counter-stain safranin
Why is it important to not completely seal the inoculated plates with adhesive tape and to incubate the plates at a lower temperature, usually 25’C?
Human pathogenic bacteria grow best under anaerobic conditions at 37’C
Factors affecting growth
- Nutrients
- Temperature
- pH
- Oxygen
How do nutrients affect growth?
Microorganisms require:
•Source of carbon, nitrogen and phosphorus
•A respiratory substrate (glucose)
•Vitamins and minerals to act as coenzymes
•Water to carry out metabolic reactions
How does temperature affect growth?
- Growth is coordinates by enzymes
- Enzymes work most efficiency over a narrow range of temperatures
- Too low - rate of enzyme-catalysed reactions becomes to low to sustain life
- Too high - denaturation of enzymes causes cell death
Mesophiles optimum temperature
20-45’C
Thermophiles optimum temperature
Above 45’C
Psychrophiles optimum temperature
Below 20’C
How does pH affect growth?
- Enzymes only work efficiently within a narrow range of pH
- For most - ph5-pH7.5
- Microorganisms can tolerate a wider range of pH than plant and animal cells
How does oxygen affect growth rate?
- Obligate aerobes require oxygen for metabolism at all times
- Obligate anaerobes find oxygen toxic and cannot grow in its presence
- Facultative anaerobes can grow rapidly in presence of oxygen but can also survive without it (slower growth rate)
What is serial dilution? (General)
- Pure cultures of microorganisms contain too many cells to allow an accurate count to be made
- Original culture is diluted down, usually in ten-fold steps
- Provides a final number within a countable range
What is serial dilution (process)
- 1cm3 of original culture is transferred to 9cm3 of sterile nutrient medium/sterile water
- Mixed to ensure an even distribution (10^-1 dilution)
- 1cm3 of mixture is transferred into 9cm3 of sterile nutrient medium and mixed (10^-2 dilution)
- Repeated a number of times to produce further 10-fold dilutions
- In order to carry out a viable count, a known volume of each bacterial culture, usually 1cm3 or 0.1cm3 is added to agar plates and incubated
What are gram-positive bacteria?
Have a thick cell wall consisting of peptidoglycan
What are gram-negative bacteria?
Have a thin layer consisting of peptidoglycan covered by a layer containing lipopolysaccharides
What do lipopolysaccharides do?
Provide some protection against antibiotics and lysozyme, making gram-negative bacteria more difficult to kill
Why do gram-negative not retain crystal violet?
- Acetone removes lipopolysaccharide membrane
- This washes crystal violet stain from the cell
- This exposes the inner peptidoglycan layer which statins fed with safranin
Why might numbers of colonies be converted to logarithms before being plotted on a graoh?
- Large numbers/large range of numbers
- Would be difficult to plot
- Log scale increases by a factor of 10 for each number
