Microbiology Flashcards
What shape are coccus bacteria? Give an example
Spherical
Eg staphylococcus, streptococcus
What shape are bacillus bacteria? Give an example
Rod-shaped
Eg E. coli
What shape are spirillum bacteria? Give an example
Spiral
Eg vibrio cholerae
By which 2 ways can bacteria be classified?
- Shape
- Grouping patterns
How can bacteria be distinguished from each other?
By their metabolic features or by antigenic features
What is meant by autotrophic bacteria?
Bacteria that synthesise cell constituents using carbon dioxide as the carbon source
What is meant by photoautotrophic bacteria?
Either photosynthesise with chlorophyll as an electron donor or alternatives using sulphur or hydrogen gas as an electron donor
Describe the Gram stain technique
- Apply crystal violet
- Apply Grams iodine solution
- Alcohol wash (decolourisation)
- Apply Safranin (counter stain)
What colour is a Gram positive bacteria after Gram stain?
Purple
What colour is a Gram negative bacteria after Gram stain?
Pink/red
Describe a Gram positive bacterial cell wall
- No outer lipopolysaccharide layer
- Thick peptidoglycan cell wall
Why do Gram positive bacteria appear purple after a Gram stain?
Thick peptidoglycan cell wall, but no outer lipopolysaccharide layer. They therefore retain the initial crystal violet stain when washed with alcohol.
Describe Gram-negative bacterial cell walls
- Have an outer lipopolysaccharide layer
- Thin peptidoglycan cell wall
Why do Gram-negative bacteria appear red after a Gram-stain?
Thin peptidoglycan cell wall and outer lipopolysaccharide membrane. When washed with alcohol, they lose this outer layer with the crystal violet stain, appearing colourless. They are then able to take up the counter stain safranin and appear red under a microscope.
What is a consequence of Gram negative bacteria having a more complex cell wall?
They are not susceptible to some antibiotics such as penicillin or lysozyme
How do bacteria reproduce?
Binary fission
Unicellular yeast may reproduce by budding
What temperature to microorganisms require for growth?
Bacterial metabolism is enzyme regulated, with most bacteria thriving between 25 C and 45 C. The optimum temperature for mammalian pathogens is 37 C (human body temp)
Microorganisms require nutrients for growth. How and what nutrients are supplied?
Nutrients are supplied in nutrient media such as nutrient agar or liquid broth. The carbon source is usually glucose, while nitrogen for amino acid and nucleic acid synthesis is provided as nitrate ions
What is the optimum pH for growth of microorganisms?
Bacteria tend to favour slightly alkaline conditions, while most fungi thrive in neutral to slightly acidic environments
What is the oxygen requirement of obligate aerobes?
They can only survive and metabolise in the presence of oxygen. They cannot survive without it.
What is the oxygen requirement of obligate anaerobes?
Can only survive and metabolise in the absence of oxygen.
What is the oxygen requirement of facultative anaerobes?
They metabolise better in the presence of oxygen but can also survive and metabolise without it
Clostridium perfringens are obligate anaerobes that cause gas gangrene. Treatment can involve the use of a hyperbaric oxygen chamber with air pressure 2.5x higher than atmospheric pressure. How would this treatment work to improve the patients health?
Oxygen would inhibit the metabolism and growth of C. Perfringens. Patients immune system can kill any current bacteria. Less toxins produced - patients begin to improve.
What happens during the lag phase of population growth?
No/little cell division. Intense metabolic activity such as enzyme synthesis.
What happens during the log phase of population growth?
Rapid increase in numbers, no limiting factors to growth. Cell division > death rate. Plenty of available glucose.
What happens during the stationary phase of population growth?
Limiting factors prevent further growth of the population eg competition for glucose. Carrying capacity has been reached.
What happens during the death phase of population growth?
Limiting factors cause the population size to decrease. Death rate > cell division. This could be due to the build up of toxic waste. Or no glucose left in the medium.
State 4 aseptic techniques which prevent contamination of pure cultures by environmental microbes
- Sterilise all media and equipment (eg inoculating loops) before use
- Handle cultures carefully, flaming the neck of culture bottles before opening and closing
- Use a lit Bunsen burner to create a convection current
- Disinfect workbenches beforehand
State 4 aseptic techniques which prevent contamination of the environment by cultures being grown
- Sterilise all work surfaces before and after experiments using a disinfectant
- Lift agar dish no more than 45 degrees
- Seal agar dishes with adhesive tape but not all the way around
- Flame the neck of the culture bottle without placing the cap on the work surface
Describe how to inoculate an agar dish (including aseptic techniques)
- Pass the metal inoculating loop through a flame until red hot, then allow it to cool
- Hold the bacterial culture in one hand then remove the cap with the little finger of the other hand without placing it down
- Flame the neck of the culture bottle for 2-3 seconds and dip the inoculating loop into the bacterial culture
- Lift the petri dish lid to 45 degrees, allowing entry of the inoculating loop, and streak the agar with the bacterial culture
- Secure the Petri dish with adhesive tape, but do not seal completely to avoid anaerobic conditions that could promote pathogenic growth
- Incubate at a suitable temperature (25 or 37 degrees Celsius) for 24-48 hours
- Sterilise all equipment after use
How does an autoclave sterilise equipment? (Glassware and metal equipment)
- Seal in an autoclave bag
- Heat to 121 degrees Celsius in steam
- Apply high pressure for 15 minutes
How is plastic equipment sterilised?
Gamma irradiation
What is the difference between the total cell count and the total viable count?
Total cell count is living and dead cells in a bacterial sample whereas total viable count is living cells in a known volume of liquid medium
How do you count the number of cells in a yeast culture using a haemocytometer?
Count a cell if it is in a square or touches the left or upper line
What does serial dilution assume?
A single bacterial cell will reproduce asexually to form a visible colony. Number of colonies therefore represents the number of bacteria in the original sample.
Describe how to carry out a serial dilution (total viable count technique)
- Place 9cm3 of sterile distilled water into 5 sterile test tubes using a sterile pipette
- Place 1cm3 of the original bacterial culture into the first tube and gently mix. The bacterial culture has now been diluted 10 times
- Transfer 1cm3 of this 10-1 dilution from the 1st tube into the 2nd tube and gently mix with 9cm3 of sterile distilled water. Bacterial culture has now been diluted 100 times (10-2) repeat this procedure for the remaining tubes.
- Transfer 1cm of each diluted sample onto a sterile nutrient agar plate. Use a sterile spreader to distribute the sample evenly on each agar plate
- Repeat twice more to give a total of 3 plates per dilution
- Seal each agar plate with tape (not all the way round) and incubate at 25 degrees C for 24-48 hours
- After incubation, identify the dilution with distinct, non-merging colonies. Count the number of distinct colonies on each plate
- Multiply the number of colonies by the dilution factor to give the number of bacteria in the original 1cm3 bacterial culture sample
What inaccuracies may occur when carrying out a serial dilution if the original bacterial culture is under diluted?
Colonies might merge and counting may be inaccurate, resulting in an underestimate of cell numbers
What inaccuracies may occur when carrying out a serial dilution if the original bacterial culture is over diluted?
If dilution is too great then there will be too few colonies on each plate to count to be statistically sound, leading to inaccuracies
Describe how you could indirectly count cells
Use a colorimeter to measure the turbidity of a culture as cell numbers increase. Bacterial population measurements are obtained by finding the suspension’s absorbance value and referencing a standard graph of light absorbance plotted against the number of bacterial cells. This results in a total cell count as the colorimeter cannot differentiate between living and dead cells
