Microbiology Flashcards
Give the structure of a prokaryote?
- No membrane bound nucleus – have a large circular piece of DNA in the nucleoid, with many small DNA fragments called plasmids.
- No membrane bound organelles.
- Smaller ribosomes than eukaryotes.
- Peptidoglycan cell wall.
- Reproduce rapidly by binary fission but also ‘swap plasmids’ to give variation.
- Size: 0.5–5 UM
What is the function of the plasma membrane?
A barrier between the environment and cytoplasm, controlling the entry and exit of substances into and out of the cell.
Approximately 7NM
What is the function of circular DNA?
To contain genes necessary for the normal functioning of the bacteria
What is the function of the cell wall?
Prevents lysis in hypotonic solution
What is the function of the capsule?
Outer layer of micro polysaccharide slime which glues bacteria together, sticks them to surfaces and protects the bacteria from attack from other cells.
What is the function of ribosomes?
Protein synthesis
What is the function of the pili?
Attaching to surfaces and for transferring plasmids by conjugation
What is the function of the flagellum?
For motality
What is the function of plasmids?
It’s circular DNA which contains extra bacterial genes including genes for antibiotic resistance. Can be exchanged between bacteria during conjugation allowing the spread of antibiotic resistance.
How can bacteria be distinguished?
By their size, shape, staining characteristics and their metabolic, antigenic and genetic features.
What is the shape of the bacteria due to?
The rigid cell wall which has a unique structure: it contains a 3-D mesh of peptidoglycan.
What is the name given to rod shaped bacteria?
Bacillus
What is the name given to the spherical bacteria?
Coccus
What is the name given to the spiral bacteria?
Spirilium
What is the arrangement when cells remain attached in pairs?
DIPLO -
e.g diplococcus
What is the arrangement when cells remain attached in chains?
STREPTO -
What is the cell arrangement when cells are arranged in squares?
TETRADS
What is the cell arrangement when there are random planes of division resulting in sheets in clumps of cells?
STAPHYLO
Why is it useful for classifying bacteria via Gram stain?
It allows microbiologist to distinguish between gram-positive and gram-negative bacteria. The different staining properties are due to the different chemical compositions of the cell wall. Before staining the bacteria is colourless, after staining the gram-positive is violet and the gram-negative is red/pink.
Describe the structure of the cell wall?
It has peptidoglycan cross linkages providing strength and maintaining support. It protects from osmotic lysis and swelling.
Describe the gram-positive cell wall structure?
It has a basic cell wall structure. It has a thick layer of peptidoglycan (thicker than negative). It has no lipopolysaccharide layer so the bacteria is more susceptible to antibiotics and penicillin and lysozyme (than gram-negative)
Describe the gram negative cell wall structure?
It has a basic cell structure with an additional outer layer of lipopolysaccharide. It has a thin peptidoglycan cell wall and a thick lipopolysaccharide layer.
Describe the staining of a gram-positive bacteria?
The crystal violet binds to the peptidoglycan so the bacteria stains purple. It retains the crystal violet/iodine complex within the cells when washed with alcohol. The dye is not washed off with ethanol so stains purple.
Give examples of gram-positive bacteria?
Bacillus, staphylococcus (MRS), strephococcus
Describe how lysozyme affects the cell wall?
Bacteria constantly makes and breaks chemical links within their cell walls. The anti-bacterial enzyme lysozyme (found in tears and saliva) hydrolyses the bonds holding the peptidoglycan molecules together.
How does penicillin affect bacteria?
It affects gram-positive bacteria as it prevents bonds from interlinking with the peptidoglycan molecules from forming. It makes a cell wall structurally weak and prone to collapse. Water uptake from osmosis bursts to cells,
Describe the staining of a gram negative bacteria?
On treatment with alcohol they lose their life a polysaccharide membrane with a thin inner peptidoglycan layer being.Therefore the crystal violet/iodine complexes is washed off along with the outer membrane. They stain red with the counterstain safranin.
Give examples of gram negative bacteria?
Salmonella species, E-coli
Why are gram negative not affected by lysozyme and are resistant to penicillin?
The outer like a polysaccharide protects the peptidoglycan below so isn’t affected by lysozyme and resistant to penicillin. Vancomycin is used which interferes with the cells ability to make proteins.
How do microorganisms reproduce and replicate?
They undergo binary fission and reproduce rapidly when in a suitable environment. In optimum they divide every 20 minutes. Bacteria can be grown on a wide variety of substrate providing that they are supplied with nutrients, water and suitable physical conditions.
What conditions are needed for growth?
Nutrients, vitamins, temperature, pH and oxygen
How are nutrients applied and its importance?
They are supplied in a nutrient media (nutrient broth) where bacteria is cultured in a liquid medium or solid medium containing agar. The media provides water and they include:
- A carbon and energy source usually glucose and stem molecules for growth.
- Nitrogen source – to manufacture proteins, nucleic acids and other nitrogenous compounds.
Some need specific vitamins or minerals.
Why are vitamins needed for growth?
As all organisms need a selection of mineral ions.
They may need vitamins and essential amino acid which they can’t make.
Why is temperature needed for growth?
The rate of enzyme activity depends on temperature as bacterial metabolism is regulated by enzymes. The range is 25 to 45°C which is the most suitable for bacteria.
Mammalian pathogens optimum is 37°C (the average human body temperature evolved and optimum to where they live)
Why is pH needed for growth?
The rate of enzyme activity depends on pH as a charge on the active site is only the ideal charge and shape at a certain pH.
Most bacteria favour slightly alkaline conditions.
What are obligate aerobes?
Bacteria which can only divide/metabolise in the presence of oxygen.
What are obligate anaerobes?
Bacteria which can only divide in the absence of oxygen.
What are facultative anaerobes?
Bacteria which can survive in the absence of oxygen that grow and divide faster in the presence of oxygen.
What is the optimum for saprotrophs?
lower temperatures
What temperature the psychrophiles grown to?
down to 15 degrees Celsius
What is the optimum of thermophiles?
At around 80 degrees celsius
What is a defined medium?
It only contains known ingredients
What is a undefined medium?
It contains unknown components
What is a selective medium?
It allows certain bacteria to grow.
Why is bacteria culture in media?
To provide the cell with all its nutritional and physical requirements.
What does aseptic technique prevent
- Contamination of the environment by the microbes being handled.
- Contamination of microbial cultures by unwanted microbes in the environment.
How do you prevent contamination of pure cultures and apparatus by bacteria from the environment?
- Sterilise all apparatus and media before used to prevent initial contamination.
- Flame necks of culture vessels before opening and closing.
- Use sterile loops to prevent subsequent contamination.
How do you prevent contamination to the environment by the bacteria?
- Sterilise the work surface before and after experiment with disinfectant.
- When carrying out inoculation:
- Hold the culture bottle (McCartney caps) in one hand, don’t put the caps on the table. Avoid contamination to and from surfaces. When flaming the neck it creates a convection current from the broth.
- Flame the neck for 2 to 3 seconds.
- Pass the inoculating loop through a blue flame until red hot and allow to cool in the air to kill any bacteria or resistant spores.
- Ethanol is used for glass spreaders which is burnt off.
- Petri dishes opened at a small angle – reduces the risk of airborne contaminants falling onto the culture, only the loop can enter.
- The petri dishes are secured with 2 to 4 pieces of tape don’t seal the whole way round as that would create anaerobic conditions which encourages pathogenic microorganisms.
- Incubate for 24–48 hours at 25°C not 37°C as that’s the ideal growth for pathogens.
- Don’t open petri dishes after incubation.
- Autoclave all glassware at 121°C for 15 minutes (15 ATM) which kills all microbes and resistant spores.
- After use disposable materials like plastic petri dishes are irradiated by the manufacturer and arrive sterile, they can be put into biohazard bags autoclaves and then put in the bin.
Why is it important to estimate population growth?
- Environmental health monitoring.
- Water supply – drinking water is not contaminated at the end of the purification process.
- Monitoring growth in fermenters – bacteria are grown in fermenters and produce various products.
How can you directly count cells?
Using serial dilution - viable cell counts
Haemocytometer - total cell counts (alive and dead)
How are viable cells counted?
Using serial dilution and plating and counting colonies
What is the assumption made when directly counting?
That a single bacteria incubated will develop into a colony (one cell – one colony)
What is a haemocytometer?
It is a type of microscope slide which is used to count individual cells. Living in dead cells can’t be distinguished unless they have been stained differently.
What are the advantages of haemocytometer?
It is quick (done in minutes), can be used to count several types of cells.
What are the disadvantages of haemocytometer?
It is difficult to count clamps (underestimate), doesn’t always discriminate between living and dead, difficult to count under a microscope, need an oil immersion lens.
What is serial dilution?
It’s when cells in a sample are counted.
Population density will still be high, so cell counts are made in known dilutions of the culture usually in tenfold steps (known as serial dilution).
Give me the serial dilution experiment?
- Used on liquid bacterial culture medium.
- 9 cm³ of sterile water is placed into six sterile tubes. - Using a sterile pipette add 1 cm³ of culture to the first tube (1 in 10 dilution).
- Using another sterile pipette transfer 1 cm³ from the first tube to the second (1 in 100 dilution).
- Continue for all 6.
- Using a sterile pipette transfer 0. 5 cm³ of each sample to a sterile agar plate.
- Using a glass spreader and aseptic technique to spread over the plate.
- Repeat so that you have three plate plates for each dilution.
- Seal with tape.
- Incubate at 25°C for 24–48 hours.
- Colonies will grow, choose a plate with a reasonable amount of colonies (50 – 100).
- Count the colonies on the three plates and calculate a mean.
- Calculate the original number of microbes by taking the number of colonies x dilution factor x 2.
What is the assumption made about serial dilution?
One colony was derived from one cell which has undergone binary fission.
What is the problem of serial dilution?
If the dilation is insufficient clumping will occur and counting becomes inaccurate. If the dilution is too great the count would be unrepresentative/unreliable.
What are the advantages of serial dilution?
Count living cells
What are the disadvantages of serial dilution?
Possible to underestimate if clamping occurs, takes too long (24 hours to see colonies), if the culture is mixed it may take more time than others.
How is growth measured indirectly?
Using the turbidimetry
How are cells indirectly measured?
A colorimeter is used to measure the cloudiness/turbidity of the culture as cell numbers increase. To find the population the suspension of absorbance is measured. It’s read off a light absorbance and number of bacterial cell graph. The result is the total cell count because the colorimeter can’t distinguish between living and dead selves.
