3.4 Microbiology Flashcards
What are some ways bacteria can be distinguished from each other?
Size
Shape
Staining characteristics
Metabolic features
Antigenic features
Genetic features
Shape of coccus bacteria
Spherical
Shape of bacillus bacteria
Rod shaped
Shape of spirillum bacteria
Spiral shaped
Pleural terms for
• coccus
• bacillus
• spirillum
• cocci
• bacilli
• spirilla
What is peptidoglycan?
Cell wall of a bacteria which is a mixture of polysaccharide and polypeptide
What is the other word for peptidoglycan?
Murein
Describe gram positive bacteria
• stains purple/violet
•thick peptidoglycan layer which retains the crystal violet
• no lipopolysaccharide layer
• not affected by lysozyme or penicillin
Describe gram negative bacteria
• stains red
• thin peptidoglycan layer
• alcohol washes the crystal violet out
• counterstain with safranin
• extra lipopolysaccharide layer
• not affected by lysozyme or penicillin
describe the gram stain procedure
- Small sample of bacteria is transferred to glass microscope slide using an inoculating loop. Slide is passed through bunsen flame a few times to fix bacteria to the slide
- Few drops of crystal violet is added and left for 30s
- Rinse excess using water
- Gram’s iodine is added for 1min to fix stain
- Purple stained bacteria are gram-positive
- Wash with alcohol for 30s to dissolve lipids in lipopolysaccharide layer and so exposing inner peptidoglycan layer
- Re-stain using safranin which stains unstained bacteria red
In what equipment is bacteria commonly cultured
Petri dish or in a flask
Whats the most common medium that bacteria is cultured in
Agar
What does agar contain in order to be a growth medium for bacteria?
Source of carbon
Source of nitrogen
Water
Vitamins and mineral salts
Suitable temperature
Suitable pH
Oxygen may or may not be required
Why is bacteria cultured at 25 degrees Celsius not 37?
To avoid growing human pathogens as they can form colonies at 37 degrees Celsuis
What are the three different oxygen requirements a bacteria can have?
Obligate aerobes
Facultative anaerobes
Obligate anaerobes
What is are obligate aerobes?
Microbes that require oxygen to carry out metabolism and reproduction
What are facultative anaerobes?
Microbes that carry out metabolism and reproduction better with oxygen but can grow without it
What are obligate anaerobes?
Microbes that cannot survive in the presence of oxygen
Obligate anaerobes in test tube
Collect at top of test tube
Facultative anaerobes in test tube
Across the whole tube but more towards the top
Obligate anaerobes in test tube
Towards bottom of test tube
What is aseptic technique?
good laboratory practice that maintains sterile conditions and prevents contamination.
What is the aim of aseptic technique
• prevent contamination of the environment by microbes
• prevent contamination of microbial cultures by unwanted microbes of the environment
What is done before an experiment to carry out aseptic technique?
• wash hands
• sterilise benches using disinfectant
• tie hair back due to bunsen burner
• sterile equipment eg petri dish(heat), inoculating loop (heated until glowing red), other equipment(autoclave)
What is done during serial dilution to carry out aseptic technique?
• lids of agar bottles held in crook of little finger - not on bench
• necks of agar bottles are flamed on opening and closing
• loops are sterilised and cooled before insertion to cultures and sterilised after
• lids of petri dishes are tilted not lifted off when agar is poured in or while plate is being inoculated
What is done after serial dilution to carry out aseptic technique?
• petri dishes are sealed with cross of tape to discourage growth of human pathogens
• plates are incubated upside down at 25 degrees Celsius for 24 hours
• plates are not reopened
• plates are autoclaved after observations are made
Total count method
Count of both live and dead bacteria
Viable count method
Count of live bacterial colonies
Methods in which total count method can be used
Haemocytometer slide
Measure colour of broth in a colorimeter
Describe how viable count technique is used to monitor population growth
• assume that one cell gives rise to one colony
• use of aseptic technique
• use of serial dilution - culture is diluted by ten-fold steps
• 1cm3 of original sample added to 9cm3 of sterile water
• these are mixed and the process is repeated
• known volume of microorganisms are added to agar plates
• these are incubated at 25 degrees C for 24-28 hours
• number of colonies is counted and multiplied by dilution factor to calculate number of cells per cm3 in original sample
How to know which plate to chose from serial dilution
One with the largest number of distinct colonies
• clumping - cannot count accurately
• too fee - inaccurate representation
What is assumed during viable count technique and serial dilution
Each colony has arisen from a single bacterium
Lag phase of bacterial growth
Slow population growth
Bacterial cells take up water, protein synthesis, DNA/RNA synthesis, enzyme production
Log phase of bacterial growth
Doubling per unit time by dividing asexually
Optimum conditions
Eventually carrying capacity is reached
Stationary phase of bacterial growth
Death rate and reproduction rate equilibrate
Limiting factors - nutrient depletion and waste accumulates
Death phase of bacterial growth
Death rate exceeds birth rate
Shortage of nutrients
Lack oxygen
Accumulation of toxic waste
Phases in order of bacterial growth
Lag
Log
Stationary
Death
Phases of bacterial growth
Lag
Log
Stationary
Death
