4 - Microbiology (C1) Flashcards
What does aseptic technique prevent?
- Contamination of the environment by the microbes being handled
- Contamination of microbial cultures by unwanted microbes from the environment
- Contamination of the experimentor
What is the function of the plasma membrane of prokaryotes?
- Barrier between environment and cytoplasm
- Controls entry and exit of substances in and out of cell
What is the function of the peptidoglycan (murein) cell wall of prokaryotes?
Prevents lysis in a hypotonic solution
What is the function of the capsule of prokaryotes?
- Outer later of mucopolysaccharide slime which can glue bacteria together and stick them to surfaces
- Protects the bacteria from attack by other cells
What is the function of the pili of prokaryotes?
Attaches to surfaces and transfers plasmids by conjugation
What is the function of the plasmid of prokaryotes?
- Circular DNA which contains extra bacterial genes for antibiotic resistance
- Can be exchanged between bacteria during conjugation allowing the spread of antibiotic resistance
What shape is bacillus?
Rod shaped
What shape is coccus?
Sphere
What shape is spirillum?
Spiral
What are the metabolic requirements for microbes?
- Suitable temp
- Suitable pH
- O2 requirements - controls growth of pathogenic bacteria which grow at a faster rate in anaerobic conditions
- Carbon source - provides energy
- Nitrogen source - required for synthesis of proteins, amino acids, DNA and RNA
- Growth factors e.g. vits and mins - essential for growth
What are some important parts of aseptic technique?
- Autoclave all glassware at 121 degrees for 15 mins
- Open petri dish at a small angle
- Keep a roaring blue flame on the bench
- Keep McCartney caps in hand and flame neck of bottle
- Flame loops, wires (until red hot) and glass spreaders (using ethanol) to red heat in a Bunsen flame
Why during aseptic technique must all glassware be autoclaved at 121 degrees for 15 mins?
To kill bacteria and spores
Why during aseptic technique must petri dish lids be opened at small angles?
To prevent bacteria entering petri dish
Why during aseptic technique must a blue flame be kept roaring on the bench?
To create a convection current to uplift air away from cultures
Why during aseptic technique must McCartney caps be kept in hand?
To not contaminate the desk
Why during aseptic technique must the neck of McCartney bottles be flamed?
To create a convection current to lift air from broth
Why during aseptic technique must loops, wires and glass spreaders be flamed?
To kill bacteria
How is a viable count of cells done?
Serial dilution is used and colonies are grown on agar plates and counted
How is a total count of cells done?
Serial dilution is used, and a haemocytometer is used to count the cells
What is the difference between a viable count and total count of cells?
Viable - only living cells are counted
Total - both living and dead cells are counted
What are advantages and disadvantages of viable counts of cells?
+ Counts living cells
- Long process
- If culture is mixed some cells may take longer to grow than others
What are advantages and disadvantages of total cell counts?
+ Quick
+ Can be used to count several types of cells
- Difficult to count if they clump
Why are bacteria cultures diluted before being counted?
As otherwise there will be too many to count
Why would it be inaccurate to choose a plate of bacteria to count with less than 50 colonies?
Unrepresentative, unreliable result
Why would it be inaccurate to choose a plate of bacteria to count with more than 100 colonies?
Clumping would occur so individual colonies wouldn’t be able to be identified
Why does the stationary phase occur in bacterial growth?
- Population has reached its maximum size
- Division rate = death rate
- Lack of nutrients or too many toxins
- Unlike wild populations, they DON’T have a carrying capacity and aren’t controlled by density dependent factors
Why during aseptic technique should cultures be incubated at 28 degrees not 37 degrees?
As this would provide optimum conditions for the growth of a pathogen
Why during aseptic technique should the lids of petri dishes be secured with 2 strips of tape but not completely sealed?
To provide the microorganisms with some O2 to grow, otherwise it would provide anaerobic growth conditions for pathogens
Why during aseptic technique should the bench be cleaned with Virkon before and after the experiment?
- Before: to prevent bacteria on the bench contaminating the microbes being handled
- After: to kill microbes used in the experiment that have contaminated the bench
What is the process of serial dilution plating?
- Add 9cm3 sterile water to 6 sterile tubes
- Swirl the culture tube to mix the contents and using a sterile syringe add 1cm3 from this tube to the next
- Continue until all 6 tubes have been done
What is the process of serial dilution?
- Using a sterile syringe transfer 0.5cm3 of each sample onto a sterile agar plate
- Use a glass spreader and aseptic techniques to spread over the plate
- Repeat this twice to give 3 plates for each dilution
- Secure the lids of the plates with 4 strips of tape and label name of bacteria, date, dilution
- Incubate plates at 28degrees for 24-48 hours
- Once colonies have grown, choose a plate with 50-100 colonies
- Count the colonies in the 3 plates for this dilution and calculate mean
- Calculate number of microbes in original
How can you calculate the number of bacteria in the original sample from a diluted sample?
- Number of colonies x dilution factors e.g. if 1 in 1000, times by 1000
- Correct for different sample sizes, e.g. check if asked for 1cm3 or 0.5cm3
What is the process of gram staining?
- Smear bacteria onto slide and ‘heat fix’
- Apply crystal violet (purple dye)
- Apply iodine (mordant)
- Wash with alcohol (decolorisor)
- Apply safranin (counterstain)
Why in gram staining is the bacteria smeared onto a slide and heat fixed?
To prevent it being washed off by staining
Why in gram staining is crystal violet applied?
It binds to the peptidoglycan cell wall and so stain all cells purple
Why in gram staining is iodine applied?
To ensure the crystal violet fixes firmly to the cell wall
Why in gram staining is the bacteria washed with alcohol?
To remove unbound crystal violet and lipopolysaccharide:
- Gram neg- lose their stain and become colourless
- Gram pos+ remain purple
Why in gram staining is safranin applied?
- Stains gram neg- cell wall red/pink
- Gram pos+ remain purple
After being gram stained what colour are gram positive+ bacteria?
Purple
After being gram stained what colour are gram negative- bacteria?
Red
What is the study of small organisms, including bacteria, important for?
- Medicine / healthcare
- Food industry e.g. dairy
- Water quality
What bacterial grouping is diplo?
A pair
What bacterial grouping is staphylo?
A clump / ‘stack’
What bacterial grouping is strepto?
A chain / ‘strap’
What is the cell wall chemistry of gram positive bacteria?
- Thick peptidoglycan cell wall (penicillin disrupts cross links + causes cell to burst by osmosis = lysis)
- Stain with crystal violet and turn purple
What is the cell wall chemistry of gram negative bacteria?
- Thin peptidoglycan cell wall
- Protective outer layer of lipopolysaccharide
- More difficult for antibiotics, enzymes and antibodies to control them
- Stain purple but when washed with ethanol, purple outer coat is dissolved, producing clear cells
- When a 2nd (counter stain) of safranin is added, the newly exposed peptidoglycan stains red
What are the names of bacteria that can survive at extremely high temperatures?
Thermophiles
What does penicillin do to gram positive bacteria?
- Prevents the bonds inter-linking peptidoglycan molecules from forming
- This is significant when the bacteria make new cell walls when they divide
- It makes the cell walls weak, and water uptake by osmosis bursts the cell
- Gram neg- bacteria are unaffected as they have a protective outer lipopolysachharide layer in their cell walls
Why are human cells not harmed by penicillin?
As animal cells don’t have cell walls
As gram negative bacteria are resistant to penicillin, how can their number be controlled?
With antibiotics that interfere with the cell’s ability to make proteins
What does a ‘defined’ medium contain?
Only known ingredients
What does an ‘undefined’ medium contain?
Components that are not all known
What does a selective medium contain?
Only components that allow certain bacteria to grow
