Component 1: Microbiology Flashcards
Draw and label the structure of a prokaryotic cell
Flagellum Pilus Circular DNA Plasma Membrane 70s Ribosome Murein Cell Wall Slime Capsule Plasmid
What is the function of the plasma membrane in a prokaryote?
- barrier between the environment and the cytoplasm
- controls entry and exit of substances into and out of the cell
What is the function of the murein cell wall in a prokaryote?
- prevents osmotic lysis
- limits pressure inside cell
- rigidity and cell structure
What is the function of the circular DNA in a prokaryote?
contains genes necessary for protein synthesis of the prokaryote
What is the function of the slime capsule in a prokaryote?
- can glue bacterium together
- allows bacterium to stick to surfaces
- protect against other cell attack
- prevents cell drying out
What is the function of 70s ribosomes in a prokaryote?
synthesis of proteins
What is the function of pili in a prokaryote?
- attaching to surfaces
- transferring plasmids (genetic information) by conjugation
What is the function of the flagellum in a prokaryote?
for motility (movement)
What is the function of the plasmic in a prokaryote?
- contains extra bacterial genes, e.g. bacterial resistance
- can be exchanged between bacteria during conjugation allowing spread of resistance
What are the 3 bacterial shapes?
- Bacillus (Rod)
- Coccus (sphere)
- Spirillum (Spiral)
What are the 4 bacterial arrangements?
- Diplo (cells are attached in pairs)
- Strepto (cells are attached in chains)
- Tetrads (cells are arranged in square)
- Staphylo (sheets and clumps)
What are prokaryotes?
- no membrane bound nucleus
- no membrane bound organelles
- small ribosomes (70s)
- cell wall made up of peptidoglycan
- small cells, 0.5-5um
- reproduce by binary fission
What is the structure of gram positive bacteria?
- cell wall has a thick layer of peptidoglycan and a plasma membrane
What happens to gram positive bacterium in the Gram stain?
- peptidoglycan holds onto crystal violet dye
- dye is not washed out by ethanol and therefore appears purple in the Gram stain
What are some examples of Gram positive bacterium?
- Staphylococcus (MRSA)
- Streptococcus
What is the structure of Gram negative bacterium?
- thick outer layer of lipopolysaccharides with a thin layer of peptidoglycan
- plasma membrane beneath
What happens to Gram negative bacterium in the Gram stain?
- crystal violet dye is removed when rinsed with ethanol (dissolves the stained lipopolysaccharide layer)
- peptidoglycan is stained with the counter-stain and therefore appears pink in the gram stain test
What are some examples of Gram negative bacterium?
Salmonella and E.coli
What antibiotics interfere with Gram positive bacterium?
- Penicillin
- prevents bonds interlinking peptidoglycan forming
- when the bacteria divide, the cell walls are weak and collapse
- water uptake by osmosis bursts the cell
What antibiotics interfere with Gram negative bacterium?
- Require antibiotics that interfere with the cell’s metabolism/protein synthesis
- e.g. vancomycin
- penicillin is not effective as the outer layer protects the peptidoglycan
Why are animal cells not damaged by penicillin?
Animal cells do not have a cell wall, not damaged by penicillin
Definition of the Gram Stain?
A method of staining the cell walls of bacteria as an aid to their identification
What is the method for the Gram-stain test?
- create a flame-fixed emulsion of bacterial samples on a slide
- flood with crystal violet, leave for 1 min then rinse of excess with sterile distilled water
- Add lugol’s iodine solution, leave for 1 min then rinse
- Flood with decolouriser (ethanol) for 30 seconds until run-off is clear
- Counter-stain with Safranin, leave for 1 min then rinse
- Gently blot dry slide
- Observe slide under oil immersion on a microscope
How do you make a heat-fixed emulsion?
sample of a culture added to water on a microscope slide and passed through a flame to fix
Why is a heat-fixed smear prepared?
- kills cells in a life-like position
- attached bacteria to the slide and not rinsed away
Why do you flood the smear with crystal violet?
- able to identify Gram-positive bacterium
- stains/binds to peptidoglycan in gram-positive bacterium
Why do you add lugol’s iodine?
Binds the crystal violet to the peptidoglycan more strongly
Why do you add alcohol/ethanol?
- washes out unbound crystal violet and lipopolysaccharide
- declourises gram-negative bacteria
Why is the counterstain/safranin added?
- counterstains Gram-negative cells pink
- Gram-positive bacteria remain purple
Conditions necessary for culturing bacteria: temperature
- bacterial metabolism is regulated by enzymes
- suitable temperature for enzyme activity is 25ºC-45ºC
- however optimum temperature for pathogenic bacteria is 37ºC (core human body temperature)
Conditions necessary for culturing bacteria: pH
optimum pH for bacterial enzymes is slightly alkaline (6.5-7.5)
Conditions necessary for culturing bacteria: oxygen
must have the right amount of O2 that their metabolism requires (obligate anaerobes etc)
Conditions necessary for culturing bacteria: C
- a carbon source (e.g. glucose)
- bacteria are heterotrophs and a carbon source is needed for bacterial respiration
Conditions necessary for culturing bacteria: N
- nitrogen source (e.g. amino acids)
- to synthesise proteins and nucleotides
Conditions necessary for culturing bacteria: vitamins and minerals
- E.g. Ca2+, Na+, Fe2+, Mg2+ and Cl-
- vitamins and minerals are essential for growth and enzyme activity
What are the two problems when working with bacteria?
- contamination of cultures from the environment
2. contamination of the environment from the cultures
What is the aseptic technique for using glassware?
- Autoclave all glassware at 121ºC for 15 minds
- prevents contamination to the environment
- kills bacteria and spores
What is the aseptic technique for using any kind of spreaders?
- passing an inoculating loop through a roaring blue flame until red hot and passing glas spreads through a flame after being dipped in ethanol
- prevents contamination to environment and to the cultures
- kills bacteria
What is the aseptic technique when using a petri dish?
- open petri dish at a small angle
- prevents contaminations to environment and cultures
- prevents bacteria entering the petri-dish
What is the aseptic technique for all cultures being used?
- have a roaring blue flame on the bench at all times
- prevents contamination of the culture
- creates a convection current to uplift air away from cultures
What is the aseptic technique when handling bottles and caps?
- keep McCartney caps in hand and flame neck of the bottle (do not put the cap on the bench)
- prevents contamination of bench and creates a convection current to lift air from the bench
Why is sterilisation important?
So pure cultures of microorganisms can be grown
What happens in an autoclave?
- an autoclave allows a pressure of 15 atmospheres and a temperature of 121ºC to be generatures
- if this is kept up for 15 mins this kills all bacteria and the spores are boiled
What is used commercially to sterilise plastic?
gamma radiation as plastic would melt in an autoclave
Define Obligate aerobes
Bacteria that require oxygen to grow or divide
Define obligate anaerobes
Bacteria that can only grow/divide in the absence of oxygen
Define facultative anaerobes
bacteria that can grow/divide in the absence of oxygen but grow faster in the presence of oxygen
Why is it important to estimate population growth?
- Environmental Health Monitoring (e.g. to check for E.coli in sea water at bathing beaches)
- Water supply (to check drinking water is uncontaminated at the end of purification process)
- Monitoring growth in fermenters (checking for unwanted bacteria)
How is the size of a population of microbes measured?
- Directly by counting cells:
• viable counts describe living cells only
• total counts describe living and dead cells - Indirectly, by measuring the turbidity (cloudiness) of the culture)
What is the method for serial dilution?
- 9cm^3 of sterile water is placed in each of a series of 6 sterile tubes
- using a sterile pipette add 1cm^3 of culture to the first tube and then with another pipette transfer 1cm^3 from the first tube to the second
- continue until all 6 tubes have been done
- use sterile pipettes to transfer 0.5cm^3 of sample onto a sterile petri dish for each of the 6 tubes and spread with a glass spreader
- forms 6 spread/pour plates
- seal plates with 3 pieces of tape and then incubate at 25ªC for 24-48 hours
How to estimate population size from a serial population?
- after the 2 days bacteria will grow
- a dish containing 20-100 colonies that are distinct and separate is chosen and the colonies counted
- to find the total viable cell count the number of colonies is multiplied by the appropriate dilution factor
What happens if the dilution is too great?
There will be too few colonies on each plate for the count
What happens if the dilution is insufficient?
colonies merge, ‘clumping’ and counting may result in an underestimation of numbers
What is the assumption made with serial dilutions and viable counts?
separate colonies of bacteria are counted assuming that every colony has originated from one single cell
Define viable counts using serial dilution
cells in a small sample are used, made in known dilutions of the culture
How are total cells counts done?
- using a haemocytometer
- special type of microscope slide to count individual cells
- not possible to distinguish between living and dead cells so the result is a total cell count
How can growth rate be estimated?
by regularly measuring the diameter of a bacterial or fungal colony spreading from a central point over the surface of a solid growth medium
