4.4: Microbiology. Flashcards
0
Q
What is a micro-organism?
A
Organisms too small to see with the naked eye such as bacteria, fungi, viruses and protoctists.
1
Q
What is Microbiology?
A
The study of micro-organisms.
2
Q
What are some of the defining characteristics of Bacteria?
A
They have no internal membranes - No ER/mitochondria etc They have small ribosomes 70S They have cell walls made of Murein.
3
Q
What are the two different ways of classifying bacteria?
A
By their shape By gram staining.
4
Q
What are the three main bacterial shapes?
A
Coccus: Sphere shaped. (Cocci plural) Bacillus: Rod shaped (Bacilli plural) Spirillum: Helical (Spirilla plural) NB you can also get Rectangular and Star shaped bacteria.
5
Q
What are the different types of cocci and their defining features?
A
Micrococcus - a single cocci (in latin “single berry”) Diplococcus - two cocci joined together (in Latin “double berry”) Saphylococcus - an irregular bunch of cocci (in Latin “bunch of grapes”) Streptococcus - a chain of cocci (in Latin “twisted berries”) Nelsseiae - coffee-bean shape in pairs Tetrads - cocci in packets of four. Sarcinae - cocci in packets of 8,16,32 cells (cuboidal structure)
6
Q
What happens when diplococci divide?
A
They remain in pairs
7
Q
What happens when streptococci divide?
A
They remain in chains after dividing.
8
Q
What happens when staphylococci divide?
A
They divide in all planes and remain grouped together.
9
Q
What is staphylococcus aureus responsible for?
A
Causing food poisoning.
10
Q
What does MRSA stand for and what is it?
A
Methicillin-resistant Staphylococcus aureus. It causes necrotising fasciitis (it’s a flesh eating bacteria)
11
Q
Outline the rules of naming species
A
The name is written in italics (on PC) and underlined when handwritten Genus name has a Capitol letter and species name is all lower case. Bacteria often have the first genus name abbreviated e.g E. Coil.
12
Q
What does Diplococcus pneumoniae cause?
A
This causes pneumonia.
13
Q
What type of bacteria is salmonella typhi/enterica and what does it cause?
A
It is a bacillus and causes food poisoning.
14
Q
What type of bacteria is Yersinia pestis and what does it cause?
A
Bacillus and causes The Bubonic plague
15
Q
What type of bacteria is Escherichia coil?
A
It is a bacillus bacteria and allows you to digest your food. There are 10¹⁴ E.coli bacteria in your gut which is 10times more than the number of up an cells in your body.
16
Q
What does bacillus anthracis cause?
A
Anthrax
17
Q
What type of bacteria is Treponema pallidum and what does it cause?
A
Spirilla - It causes syphilis
18
Q
What type of bacteria is Borrelia burgdorferi and what does it cause?
A
Spirilla - it causes Lyme disease.
19
Q
Why do different bacteria have different shapes?
A
Because they have rigid cell walls with these unique shapes.
20
Q
What are the advantages of using shape as a means of classification?
A
It’s quick, easy and cheap. There properties are useful if you’re trying to decide what disease someone has.
21
Q
How else can bacteria be classified?
A
By their metabolic reactions.
22
Q
What are SS agar (salmonella-shigella) plates?
A
SS agar plates contain lactose, bile salts, ferric citrate and neutral red. They allow you to tell the difference between bacterial species on the basis of their metabolism.
23
Q
What is the purpose if the bile salts in the SS plates?
A
The bile salts select for Gram-Negative bacteria.
24
What is the purpouse of the other components of an SS Agar plate (all components excluding bile salts)?
The other components differentiate between different Gram Negative bacteria.
25
What are coliform bacteria?
These are rod-shaped Gram-negative non-spore forming bacteria which can ferment lactose with the production of acid and gas when incubated at 35–37°C.
26
What happens when coliform bacteria such as E.coli are grown on an SS plate?
Coliform bacteria ferment the lactose to give pink colonies. This is because they do not produce any hydrogen sulfide.
27
What happens when salmonella bacteria are grown on an SS plate?
These bacteria do not ferment lactose but do produce hydrogen sulfide gas which gives colourless colonies with black centres.
28
What happens when shigella bacteria are grown on an SS plate?
Shigella bacteria do not ferment lactose or produce hydrogen sulfide gas so the resulting colonies will be colourless.
29
What are the three types of bacteria in terms of their use/disuse of oxygen in their metabolism?
Obligate aerobes Obligate anaerobes Facultative anaerobes
30
What is an obligate aerobe?
These bacteria require oxygen for growth at all times. (Oxygen is obligatory) E.g Mycobacterium tuberculosis which causes TB.
31
What is an obligate anaerobe?
There require the absence of oxygen for growth at all times. They find oxygen toxic because it acts as a competitor to their respiratory reactions. They might respire by fermentation or by using methane as a substrate. E.g clostridium tetani (tetanus), clostridium botulinum (botulism), Clostridium perfringens (gas gangrene) and many soil bacteria.
32
What is a facultative anaerobe?
These bacteria grow rapidly in the presence of oxygen and can grow slowly in the absence of oxygen e.g E.coli can grow in your intestines where there is little/no oxygen but can also be grown in the lab.
33
What is Gram staining?
This was invented by Hans Christian Gram and was designed as a way of staining bacteria to make them easier to see but it also happens to distinguish between two different groups of bacteria.
34
How do gram positive bacteria appear after gram staining?
They retain the purple stain in their cell walls.
35
How do gram negative bacteria appear after gram staining?
They do not retain the purple stain in their cell walls and so are stained red by the counterstain.
36
How do you perform a gram stain?
1.) mount sample on a slide adding sterile saline if necessary 2.) heat the slide to kill the bacteria and make the slide safe to handle 3.) flood the slide with Crystal violet stain and leave for 1 minute - this stains all cells dark purple 4.) gently wash off crystal violet under running water 5.) flood the slide with lugol's iodine and leave for 30 seconds. This reacts with crystal violet making all the cells appear blue-black. 6.) rinse the slide gently with acetone. This washes the iodine-dye complex out of gram negative cells. 7.) counter stain the slide with safranin/eosin/fuchsin.
37
Why do gram positive bacteria retain the Crystal violet stain?
Because of the structure if their cell walls: gram positive bacteria have a plasma membrane and then a thick peptidoglycan layer on the outside and so retain the stain.
38
Why do gram negative bacteria not retain the crystal violet stain?
Because of the structure of their cell wall: gram negative bacteria have a plasma membrane and then a thin peptidoglycan layer and then a lipopolysaccharide layer.
39
Do all bacteria stain with gram stain?
No because some bacteria have a waxy cell wall that resists the stain like the mycobacteria e.g mycobacteria tuberculosis.
40
What is the best way to classify bacteria?
By DNA sequencing. This is how the three domains (bacteria, Archaea and Eukaryota) were identified.
41
How are bacteria grown in liquid cultures?
In liquid culture bacteria under the surface may not get enough dissolved oxygen so they are usually grown in shaking incubators to aerate them.
42
What do bacteria need to grow?
Oxygen/no Oxygen (depending) Water (as all metabolic reactions occur in aqueous solution) A carbon source (usually organic (glucose)) A nitrogen source (organic or inorganic) to make amino acids. A phosphorus source (to make A.T.P) A respiratory substrate (energy source (glucose again)) Other nutrients: vitamins, growth factors, minerals.
43
What is LB (Luria-Bertani) broth?
It is a mixture of chemicals which provide the nutrients necessary for bacteria to grow: In 1 litre: 10g tryptone 5g yeast extract 10g NaCl 2g glucose (1.5% agar for plates)
44
How can galactose be useful in bacterial culture?
If you want to artificially give your bacteria a new gene on a plasmid (transformation) you can tag it with a gene to use galactose as an energy source. This ensures that only the bacteria with the new galactosidase and gene of interest will survive.
45
Sometimes transformed bacteria rearrange the plasmid to keep the galactosidase marker, but spit out the gene you wanted. How can this be prevented?
By growing the transformed bacteria on an analogue of galactose: Put your gene of interest in a plasmid in such a way that it interrupts the galactosidase gene.
46
What is Tryptone?
Milk protein (mostly casein) digested by trypsin. It is a good source of amino acids and Nitrogen. It contains no carbohydrate allowing to to substitute whatever you choose.
47
What is the function of yeast extract in LB broth?
Yeast extract is ground up yeast. It is a good source of amino acids and Nitrogen and iron and vitamins.
48
What is TB (Terrific Broth)
This contains more glucose for lots of growth.
49
What is MacConkey agar?
This is for gram-negative bacteria.
50
What is blood agar?
This is used to grow bacteria that live in the bloodstream and lose red blood cells to get their nutrition. It contains horse or sheep blood.
51
What is buffered charcoal yeast extract (BYCE) agar?
These use charcoal as an alternate carbon source and are used to grow legionella bacteria.
52
What temperature do bacteria need to be at in order to grow best?
Most bacteria are mesophiles and have an optimum growing temperature of 25°C - 45°C as this is the optimum temperature of their enzymes. Some bacteria are Thermophiles and prefer higher temperatures (\>45°C) and others are psychrophiles and grow best at about temperatures \<20°C.
53
What is the optimum temperature for most human pathogenic bacteria?
Their optimum temperature is 37°C just like us which is why you run a temperature when you have an infection in order to denature the bacterial enzymes.
54
What pH do bacteria need to grow best?
Their enzymes have a optimum pH of 7.4. This is why you have stomach acid - so that bacteria are killed before they reach your intestines. Fungi prefer slightly more neutral or acidic conditions. However there are exceptions and bacteria tolerate a wider pH range than animal or plant cells.
55
What are the four growth phases a bacterial culture will go through?
Lag phase Log phase Stationary phase Death/decline phase
56
What happens in the lag phase?
In this phase: Very few bacteria are present to start and there is a slow increase in the number of cells in the population. Bacteria adjust to new conditions; switching on new genes and producing digestive enzymes to suit the environment. (As they are saprophitic)
57
What happens in the log phase?
In log phase: The rate of cell division is at it's maximum - population grows at an exponential rate. Nutrients are in plentiful supply. Waste products are only in low concentrations.
58
What happens in the stationary phase?
In the stationary phase: The number of new cells being formed is equal to the number of cells dying and equilibrium is established. Carrying capacity has been reached. Limiting factors like nutrient depletion and waste product accumulation prevent a further increase in cell numbers.
59
What is carrying capacity?
The maximum population size that a given environment can support indefinitely.
60
What happens in the death/decline phase?
This happens because of shortage of nutrients and/or the accumulation of waste products to harmful levels. In the death phase: More cells are dying than are being produced. Carrying capacity is exceeded Population begins to decrease
61
What is the purpose of Aseptic technique?
They ensure that the bacteria you want are not contaminated by unwanted bacteria from the environment and no bacteria escape to cause infections or damage to the environment.
62
What are the laboratory biosafety levels?
Category 1 Category 2 Category 3 Category 4
63
What is a category 1 lab?
In a category one lab researchers are required to wear gloves, techniques are done on the open bench and samples are decontaminated after use by autoclaving. Bacteria like non-pathogenic E.coli could be handled in a cat 1 lab.
64
What is a category 2 lab?
This is the same as a category one lab except: More advanced training is required Some aerosol generating techniques are carried out in a safety cabinet Access to the lab is limited. C.difficile, MRSA, hepatitis A/B/C, salmonella, influenza, Lyme disease can be looked at in this lab
65
What is a category 3 lab?
In a Cat 3 lab: All procedures are carried out in a safety cabinet by highly trained staff wearing protective suits. The lab has a double door entrance with positive pressure (so air is sucked into the lab instead of escaping into the environment.) Yersinia pestis, Mycobacterium Tuberculosis, SARS, rabies can be used In this lab
66
What is a category 4 lab?
Staff wear positive pressure suits with segregated air supply and work in pairs The lab has uv treatment, showers and airlock at the entrance. The lab is usually in its own separate building. A cat 3 lab is needed to look at Ebola, smallpox and unknown new bacteria and viruses.
67
How do you achieve the aims of aseptic technique?
By killing anything outside before it can get in and by stopping anything inside from getting out.
68
How is equipment sterilised for aseptic technique?
Heat treatment - autoclaving or flaming Gamma irradiation - plasticware Cleaning with disinfectants.
69
What does an autoclave do?
An autoclave will heat all glassware, metal and media (nutrient broths, agar) by pressure cooking at 121°C for \>15 minutes. This is better than boiling because it kills endospores which are resistant to boiling.
70
What is an endospore?
They are dormant capsules protecting the bacterial DNA. They are formed under unfavourable conditions and are resistant to heat, drying, pH changes and disinfectants.
71
What do you do before using bottles/tubes?
You flame the mouths of the bottles/tubes for 2-3 seconds after opening to kill any outside bacteria.
72
What do you do to all inoculation loops before use?
You flame them until they are glowing red.
73
How is plasticware prepared in aseptic technique?
Sterile Plasticware is opened and closed inside the biosafety cabinet after spraying with alcohol.
74
What must be done in preparation for aseptic technique?
All glassware, metal equipment and media (nutrient broths etc) must be autoclaved. The mouths of tubes and bottles must be flamed for 2-3 seconds to kill any outside bacteria. Inoculating loops are flamed until they glow red. Hands are washed with antibacterial soap. All surfaces are cleaned with disinfectant and ethanol before use. A lit Bunsen burner is present to provide an updraft to move airborne contaminants away from your cultures and plates.
75
What must be done during aseptic technique?
Bottle tops should be held in your hand with your little finger to keep them away from the bench surface. Do not touch the sides of any bottles or tubes with the loops or pipettes. Only open Petri dish lids just wide enough to add the sample.
76
What must be done after performing aseptic technique?
Everything has to be sterilised again after it has been touched by cultured bacteria (autoclaving, flaming etc) All plasticware is incinerated All solutions are autoclaved.
77
What happens to a plate once it has been inoculated?
The plates are sealed with adhesive tape (parafilm) leaving a gap to avoid the creation of anaerobic conditions inside the dish which would encourage pathogenic bacteria to grow. They are then incubated overnight at 25°C (to limit the growth of pathogenic bacteria which grow best at 37°C.
78
Why would you use culture techniques to estimate the number of bacteria present in a culture?
To check the levels of infection medically. To check the bacterial levels in food and restraunts To check bacterial levels in the water supply To check bacterial growth in industrial fermenters.
79
What is a direct bacterial count?
This is when you take a sample and count actual cells.
80
What is a indirect bacterial count?
This is when you use some other measure related to cell number, like turbidity (cloudiness).
81
How do you perform a direct count?
Take a sample of the culture and place it on a haemocytometer and count the number if bacteria in each grid and then work out the dilution compared to the original sample volume to work out total cell number in the original culture.
82
What is a haemocytometer?
This is like a coverslip over a grid of known size which takes a set volume of liquid.
83
What is a total cell count?
This is when you count all the cells - dead or alive - so this is an overestimation of the numbers of bacteria in your culture.
84
What is a viable cell count?
In a viable count Trypan Blue is used to stain dead cells and only living cells are counted.
85
When is an indirect count useful?
This is used for medical samples (urine) and water samples.
86
How can turbidity be measured for an indirect count?
By measuring how much light passes through a sample quantitatively by using a colorimeter or a spectrophotometer and how much is scattered by bacterial cells. You then compare your measurement of how much light gets through to a standard graph of light absorbance against known cell numbers.
87
Is an indirect count a viable count or a total count?
Total as there is no way to differentiate between live and dead bacteria.
88
What is another way of taking a viable count?
By growing a samples (after serial dilution) on a plate and counting the number of individual colonies and assuming each colony came from a single cell and then multiplying by the dilution factor. This is a viable count as only live bacteria will produce colonies.
89
What are the problems associated with taking a viable count by counting colonies in a plate.
This method assumes all cells were separate when plated so it is an underestimate. If too many bacteria are present you won't get individual colonies.
90
How do you perform a serial dilution?
This is a dilution of a sample usually performed in 10 fold steps. E.g 1ml is taken from the original sample and mixed with 9ml of water. Then 1ml of that solution is taken and mixed with another 9ml of water... A third dilution is usually performed.
91
What criteria do we look for in a plate for making a viable cell count?
Some plates will have too few colonies to be a true representation of the original culture and others will have too many bacteria and the colonies will all have merged. We look for one in between these two in the "countable range".
92
How can you make a rough estimate of growth rate?
By regularly measuring the diameter of a colony as it grows and spreads out.
93
What is biotechnology?
The industrial application of biological process.
94
What is industrial fermentation used for?
It is used to culture bacterial and fungal cells to create large numbers of cells to produce large amounts of product.
95
Define fermentation.
The chemical breakdown of a substance by bacteria, yeasts or other microorganisms.
96
Why is the use of biotechnology to create a product better than by industrial chemestry?
Microbes grow exponentially to huge numbers with only simple additions like glucose. They grow at lower temperatures than those used in industrial Chemestry - so it is cheaper. It is also more efficient due to the presence of enzymes.
97
What is a primary metabolite?
These are substances the microorganism requires for it's normal metabolism. E.g enzymes
98
What are the optimum culture conditions to produce a primary metabolite?
In order to maximise the production of substances needed for normal metabolism we aim to keep microbes growing and healthy - keeping them in the log phase. This is done in continuous fermentation.
99
How is continuous fermentation acheived?
This is used for the production of primary metabolises:- Microbes are producing enzymes to respire and maybe photosynthesise to increase their biomass. If nutrients are constantly added and products taken away then production can continue for a long time.
100
What is a secondary metabolite?
These are substances not required for the normal metabolism of the microbes which they only make under certain circumstances e.g antibiotics.
101
Why are secondary metabolites produced?
Some microbes produce antibiotics under overcrowded conditions when limiting factors kick in like shortage of nutrients in order to kill off the competition.
102
What are the optimum conditions for the production of a secondary metabolite?
In order to maximise production of substances only needed under crowded conditions we crowd the bacteria and keep them in the stationary phase at the carrying capacity. This is done in batch fermentation.
103
How is batch fermentation acheived?
This is done in a large closed tank (40-200dm³) made of stainless steel to resist corrosion from acidic fermentation products and high pressure. You need a pure culture of your microorganism in order to get a pure product during and after growth in the ferment or vessel. Everything has to be sterilised before use. Optimum conditions need to be maintained throughout the process.
104
Why might aeration be necessary?
To ensure the maximum growth rate of aerobes.
105
What is the purpose of a sparging ring?
This keeps the culture aerated and aid mixing. It is a metal ring with jets to introduce bubbles of air into the system.
106
How does mixing the culture help growth and how is it acheived?
This improves growth by improving contract with the nutrients. It is done by aeration with the sparger or using a separate mixer.
107
What is the purpose of a water jacket?
This helps keep temperature constant by cooling the culture. This is necessary because reactions create heat.
108
How is the culture's pH kept constant?
By adding a pH buffer.
109
When is penicillin produced?
Penicillin is a secondary metabolite and so is produced when the fungi reach the stationary phase - when glucose has been depleted.
110
Outline penicillin production.
1.) sterilise the fermenter vessel using high pressure steam to prevent contamination from microbes and maintain efficiency. 2.) add sterile nutrient medium 3.) add a pure culture of penicillium notatum 4.) provide the fungus with suitable conditions for growth - providing sterile air for aerobic respiration and maintaining an optimum temprature by heating and then subsequent cooling with a water jacket; Mixing and maintaining a optimum pH. 5.) allow fungus to grow and wait until stationary phase is reached (once glucose is depleted). The fungi then begin secreting penicillin into culture. 6.) drain culture after about 6 days 7.) filter to remove mycelia and then purify filtrate to extract penicillin 8.) clean and sterilise ready for next batch.
111
How does penicillin work?
Penicillin prevents the formation of cross-links between peptidoglycan units in the bacterial cell wall during division. This weakens the cell wall and leaves the bacteria susceptible to osmotic lysis. It is more effective against gram-positive bacteria because they have a thick peptidoglycan layer in their cell walls.
112
Label this fermenter diagram
