Bacteria And Disease Flashcards
What are pathogens?
Microorganisms that cause disease
What does it mean to culture microorganisms?
Microorganisms are provided with the nutrients, level of oxygen, pH and temperature that they require to grow in large numbers so they can be observed and measured
Why is it important to take care when culturing microorganisms?
- even if the microorganism you are culturing is completely harmless there is always the risk of a mutant strain arising that may be pathogenic
- there is a risk of contamination of the culture by pathogenic microorganisms from the environment
- when you grow a pure strain of a microorganism the entry of any other microorganisms from the air or your skin into the culture will contaminate it
What does sterile mean?
It is a term ised to describe something that is free from living microorganisms and their spores
What are the precautions you must take when culturing?
- All of the equipment must be sterile before tje culture is started
- once a culture has grown it must not leave the lab
- all cultures should be disposed of safely by sealing them in plastic bags and sterilising them at 121°c for 15 mins under high pressure before throwing them away
What is the nutrient medium?
A substance used for the culture of microorganisms, which can be in liquid form or in solid form
What is the nutrient broth?
A liquid nutrient for culturing microorganisms commonly used in flasks test tubes or bottles
What is nutrient agar?
A jelly extracted from seaweed and used as a solid nutrient for culturing microorganisms commonly used in petri dishes
Why is agar a useful jelly?
Because although it sets at 50°c it does melt until it is heated until 90°c
What is a selective medium?
A growth medium for microorganisms containing a very specific mixture of nutrients so only a particular type of microorganism will grow on it
What are selective medium good for?
Indentifying particular mutant strains of microorganisms, antibiotic resistant strains, and strains that are genetically modified
What is inoculation?
The process by which microorganisms are transferred into a culture medium under sterile conditions
How does inoculation take place?
- sterilise rhe inoculating loop by holding it in the Bunsen burner until it glows red hot and then leave to cool
- dip the sterilised loop in the suspension of the bacteria. Streak the loop across the surface of the agar, avoiding digging into the agar. Replacing the petri dish lif, tape closed and label. Turn dish upside down
How is the inoculating loop used?
By scraping off bacteria from onee solid media surface either into a liquid medium or streaking across another solid medium plate
How can an inoculating broth be used?
Make a suspension of the bacteria to be grown and mix a known volume with the sterile nutrient broth in the flask. The flask is then stoppered again as quickly as possible with cotton wool to prevent contamination from the air and clearly labelled. The flask is incubated at a suitable temperature and is often shaked to make sure the broth is aerated, allowing oxygen to the growing bacteria
What are ways of isolating the desired microorganism to get a pure culture?
- growing a culture under anaerobic conditions will ensure only anearobic bacteria will survive. Growing it under aerobic conditions will ensure only aerobic bacteria survive. This may not allow you to complete the seperation of microorganisms necesssry for a pure culture but it will reduce the variety
- the nutritional requirements of different organisms vary greatly. You can produce a medium that will favour the growth of the organism you wish to culture and inhibit the growth of others this allows you to identify the colony you want and then reinoculate it to produce a single pure culture.
- there are indicator media that cause certain types of bacteria to change colour
- we can only culture about 1% of the known species of bacteria
What are ways of producing a medium specific to a bacteria?
Controling the range of nutrients available or introducing selective growth inhibitors, antibiotics or antifungal chemicals
What is a haemocytometer?
A thick microscopic slide with a rectangular indentation and etched grid of lines that is used to count cells
How do you use a microscope and haemocytometer to count cells?
The sample of nutrient broth is diluted by half with an equal volume of trypan blue, a dye that stains dead cells blue so you can identify and count only the living cells. The cells are viewed using a microscope and counted.
Each corner of the haemocytometer has a square divided into 16 smaller squares. The number of cells in each of these four sets of 16 squares is usually counted and the mean calculated. The haemocytometer is calibrated so that the number of cells in one set of 16 squares equates to the number of cells x10’4 per cm3 of broth. By taking measurements at regular time intervals throughout the life of a bacterial colony, a pciture of changing cell numbers can be built up
What is turbidimetry?
A method of measuring the concentration of a substance by measuring the amount of light passing through it
What does turbid mean?
Turbid is a term used to describe something that is opaque or thick with suspended matter
How does turbidity take place?
As the numbers of bacterial cells in a culture increase it becomes more turbid. As a solution gets more turbid it absorbs more light and lets less pass through. A colourimeter measures how much light passes through the sample and therefore, indirectly how many microorganisms are present. A calibration curve is produced by growing a control culture and taking samples at regular time intervals. The turbity of rach sample is measured and a cell count using a haemocytometer is made for each sample. This gives us a relationshio between the turbidity of the culture and the number of bacterial cells present. Using this calibration curve we can measure the number of microorganisms simply using turbidimetry if we wanted to e.g. investiagte the effect of different conditions on the growth rate of the microorganism
What is dilation plating?
A method used to obtain a culture plate with a countable number of bacterial colonies
What is a total viable cell count?
A measure of the number of cells that are alige in a given volume of a culture
What is the technique of dilution plating based on?
The idea that each of the colonies on an agar plate has grown from a single viable organism on the plate
What is dilation plating used to find?
The total viable cell count
How does the technique of dilation plating work?
Often a solid mass of microbial growth is present after culturing and it is not possible to work out the individual colonies. The orignal culture is dilyted in stages until a point is reached where the colonies can be counted. If the number of colonues is multiplied by the dilution factor then a total viable cell count for the original sample can be determined. Because there are often two or more plates where counting individual cells is possible it is possible to reach a mean giving a reasonably accurate number of the cells in a sample
How can the accuracy of dilution plating be checked?
Using a haemocytometer to count the cells in the original culture
What is a sinole way to asses growth when culturing fungi and what can this be used to compare?
Measure the diameter of the patches of mycelium
This can be used to compare growth rates in different conditions
What is a good method to find the optimum temperature for the growth of a fungi?
Identical petri dishes containing indentical growth medium are inoculated with the same number of spores of a fungus. The petri dishes are cultured at different temperatures with several identical dishes grown at each temperature. After a set period of time the diameter of each fungal colony is calculated and the mean value for the diameter at each temperature is calculated. The temperature that has resulted in the largest mean diameter is the optimum temperature for growth.
Why is measuring the diameter of colonies of bacteria grown not a useful way to measure growth?
Because the microorganisms are so small the colony grows slower and so isn’t as easy to measure
What is a very effective way to discover the best array and concentration of nutrients ir the optimum pH at which to grow fungi?
Testing the dry mass of the organism
How is the technique of measuring the dry mass of an organism done?
Normally a liquid growth medium is used. Samples of broth can be removed at regular intervals and the fungi seperated from the liquid by centrifugation or filtering. The material is then dried thoroughly to the point that no more loss of mass is recorded e.g. in an oven overnight at 100°c. This gives a measure of the dry mass of biological material in a certain volume of the culture medium and an increase or decrease in the dry mass gives an indication of the increase of decrease in the mycelial mass. The conditions which produce the greatest dry mass of fungus are the optimum ones for growth
What is the time span between bacterial divisions known as?
Generation time
What are the two reasons exponential growth in a bacterial culture does not continue?
- a reduction in the amounts of nutrients available. At the start of the culture there are more than enough nutrients for all the microorganisms but as the numbers multiply exponentially in the log phase of growth the excess is used up. Unless fresh nutrients are added, the level of nutrients available will become insufficient to support further growth and reproduction and so will limit growth of the organism
- a build up of waste products. At the beginning of the growth cycle of a bacterial culture the waste products are mineral but as cell numbers rise the build up of toxic material is enough to inhibit further growth and even to poison and kill the culture. In particular as co2 produced bu the respiration of the bacterial cells builds up the pH of the colony falls to a point where the bacteria can no loner grow
Why are log numbers used to represent the bacterial population?
Because the difference in numbers from the initial organism to the billions of descendants is too great to represent using standard numbers
What are logarithms?
Powers of a base number
Why is a growth of bacteria against time graph semi logarythmic?
Because growth of the y axis is logarithmic and time is kept on a normal scale on the x axis
What is the equation to calculate the number of bacteria in a population and what does each symbol mean?
Nt = N0 x 2kt
Nt = number of organisms at time t N0 = number of organisms at time 0 k = the exponential growth rate constant t = the time the colony has been growing
How do you work out the exponential growth rate constant?
k = log10Nt - log10N0/ log102 x t
