1.2: Techniques for microbiology Flashcards
1
Q
What is phototrophs?
A
Absorption of light energy
2
Q
What are the two sources of energy living organisms can get a continuous supply from?
A
- phototrophs
- chemotrophs
3
Q
What is chemotrophs?
A
Chemical potential energy in food
4
Q
What are the 2 sources of carbon organisms get their supply from?
A
- Autotrophs
- Heterotrophs
5
Q
What are autotrophs?
A
Inorganic carbon (CO₂)
6
Q
What are heterotrophs?
A
organic compounds (e.g. sugars)
7
Q
Why do in vitro microorganisms need nutrients for?
A
- To generate ATP/energy source
- To synthesise cell materials
- must be present in the culture medium
8
Q
What must the culture medium contain?
A
all necessary nutrients in sufficient quantities
9
Q
What is a synthetic culture media?
+ uses
A
- Made from laboratory chemicals in carefully measured quantities
- for growing microorganisms when you know their exact requirements
10
Q
What is complex culture media?
+ uses
A
- contains some ingredients of unknown chemical composition
- for growing a wide range of microorganisms or when the requirements are not known
11
Q
What is selective culture media?
+ uses
A
- very specialised, suitable for growing a specific organism
- diagnostics (e.g. detecting salmonella in faeces)
12
Q
Why is carbon needed for microorganism growth?
A
To build organic molecules (e.g. proteins, carbohydrates)
13
Q
Why is nitrogen needed for microorganism growth?
A
To build protein and nucleic acid (DNA, RNA)
14
Q
Why is sulphur needed for microorganism growth?
+ source
A
To build amino acids
+ many need to be provided as sulphates
15
Q
What minerals are needed for microorganism growth?
A
- macronutrients (potassium, magnesium and phosphate)
- micronutrients (manganese, cobalt, copper, zinc, molybdenum)
16
Q
What does potassium do for microorganisms?
A
Enzyme cofactors
17
Q
What does magnesium do for microorganisms?
A
help make Chlorophyll
18
Q
What does phosphate do for microorganisms?
A
Make ATP/DNA
19
Q
Why are minerals needed for microorganism growth?
A
For enzymes to function, often act as cofactors
20
Q
Why are growth factors needed/controlled for microorganism growth?
A
- Cannot be synthesised from simpler substances
- include Amino acids, purines and pyrimidines (nucleotides), vitamins
- Needed to make proteins, DNA and cofactors for enzymes
21
Q
What are thermophiles?
A
microorganisms that have optimum temperatures at which their enzymes will function above 40 degrees
22
Q
What are mesophiles?
A
microorganisms that have optimum temperatures at which their enzymes will function between 20 - 40 degrees
23
Q
What are psychrophiles?
A
microorganisms that have optimum temperatures at which their enzymes will function below 20 degrees
24
Q
What PH do most bacteria grow best at?
A
PH 7, preferring to grow on the range 6-8
25
What PH do few bacteria grow at?
Below a PH of 4
26
What is the extreme that bacteria can grow at? specifically Thiobacillus thiooxidans
0
27
What do photoautotrophs and chemoautotrophs use as their carbon source?
Carbon dioxide
28
What do heterotrophs use as their carbon source?
Complex organic molecules, such as cellulose
29
What source does nitrogen use?
- some microorganisms can fix atmospheric nitrogen
- many take up nitrogen as nitrates
- some microorganisms need the ammonium, ion rather than nitrate
30
How are macronutrients sourced?
As salts in the culture media
31
How are macronutrients sourced?
often present in adequate amounts as contaminants (e.g. in tap water used)
32
Where are growth factors sourced?
needed in small quantities and added to the culture medium
33
What is the source of oxygen requirements?
- water in the culture medium containing oxygen
- atmospheric oxygen
34
How is temperature sourced for microorganisms?
incubator or warming/cooling jacket
35
How is PH Sourced for microorganisms?
- often produce waste products which alter the pH of the medium, buffers are added
36
What are buffers?
substances that maintain the pH
37
How do you aseptically pour an agar plate?
- hold flask top in one hand and discard plug into disinfectant
- flame mouth of flask
- mix and pour immediately into sterile Petri dishes
- allow plates to set (15 min approx.)
38
How do you create a bacterial lawn?
- sample is pipetted onto surface of agar plate (0.1ml or less)
- sample is spread evenly over surface using glass spreader
- incubate
- surface colonies
39
How do you do the pour plate method?
- sample is pipetted into the sterile plate
- sterile medium is added and mixed well with inoculum
- incubate
- should see subsurface and surface colonies
40
What are the differences between - pour plates - VS Spread plate
- obligate / facultative anaerobes / aerobes
- large volumes of inoculum
- bacteria grow on surface and subsurface colonies
- large surface area for colony growth
- don't always get an even distribution of colonies
- organisms may die if agar is too hot
- may be difficult to pick out colony growing in agar
41
What are the differences between pour plates VS - Spread plate -
- aerobic organisms
- only use small volumes of inoculum
- bacteria only grow on surface
- smaller surface area for colony growth
- more even distribution of colonies
- agar is already set, bacteria won't die
- picking a colony from the plate is less likely to disturb other colonies
42
What does pick out mean, in terms of colonies?
select colonies and grow in different media
43
What are obligate anaerobes?
microorganisms that will die in O₂
44
what are facultative anaerobes?
can survive with O₂ and can survive without
45
What is obligate aerobes?
Need oxygen to survive
46
How do you produce a streak plate?
- hold inoculating loop in flame
- remove lid from culture
- pass neck of bottle through the flame
- dip the cool loop into the broth culture
- flame neck again
- raise lid of Petri dish with other hand, only enough to allow loop inside. streak surface in three parallel lines
- resterilise loop and break as shown. sterilise loop at each corner
- seal dish with adhesive tape and incubate (don't tape fully shut - need O₂)
47
What is the purpose of a streak plate?
- isolate pure colonies with incubation due to diluted quantity of bacterial culture
- separates mixed cultures into pure ones as each colony grows from a single cell
48
How do you perform the inoculating stab technique?
- use a sterile straight wire to inoculate a stab medium
- stab through the centre of the medium taking care to withdraw the wire along the line of the inoculum without making further stab lines
49
What does the stab method used to indicate?
- motility of bacteria
- oxygen requirements of bacteria for growth
50
Bacterial growth curves - LAG phase
Growth is slow as there are only a few bacteria to divide. The microbes are adjusting to the food supply and making the enzymes needed to use the supplied nutrients
51
Bacterial growth curves - LOG phase
Maximum rate of cell division fission. Good supply of nutrients and little build-up of toxic waste
52
Bacterial growth curves - Stationary Phase
Cell division equals cell death due to competition for food and toxic waste build up. Carrying capacity has been reached
53
What is carrying capacity (bacterial growth curves)
The maximum population size the environment can sustain
54
Bacterial growth curves - Decline phase
More organisms are dying than are being produced by binary fission. This is due to exhaustion of the food supply and build up of toxic waste
55
What is the growth rate?
the increase in cell numbers over a measured period of time
56
What is generation time and when does bacteria usually multiply?
- Time taken for a bacterial population to double in number
- doubles every generation
57
What are dilution factors?
A way of expressing as a ratio the volume of the original bacterial solution in the final diluted solution
58
what methods can be used to measure growth?
- viable count
- total count
59
What do viable count include?
only counting living cells capable of dividing
60
What do the total count involve?
Counts all cells in a culture whether dead or alive
61
How do you calculate how much stock solution is needed (dilution factors)
volume of final solution / dilution factor
62
What are serial dilutions?
is the repeated step by step dilution of a bacterial culture. The dilution factor at each stage is usually kept constant.
63
What is the purpose of serial dilutions?
allow highly concentrated bacterial samples to be more easily counted
64
What are viable counts?
- a known volume of culture is serially diluted
- each dilution is grown on sterile nutrient agar
- plates are made in triplicate
- each colony produced after incubation has arisen from a single microorganism so the number of microorganisms in the original sample can be calculated
65
Why are the viable count plates in triplicate?
To get the minimum amount of bacteria for the mean and to check for anomalies
66
What are the methods of doing total counts?
haemocytometer
turbidimetry
67
What is a Haemocytometer, how is it used?
- This is a special microscope slide with a very fine grid scored in the centre.
- A cover slide is added to the slide then a drop of the sample is placed on the slide and placed under the microscopes.
- the number of microorganisms in several squares are counted and an average found
68
Why is dye used in the haemocytometer?
to be able to distinguish between dead and alive cells.
The dead cells appear blue because the dye moves into the membrane.
69
What is the size of the squares inside the centre square in the haemocytometer?
0.2mm by 0.2 mm
70
What is the size of the whole centre square in the haemocytometer?
1mm x 1mm
71
What is the depth of the haemocytometer?
0.1mm deep
72
What is the north west rule for counting cells?
only count cells which overlap the lines on the top and left NOT the bottom or right
