1. 4.4 Microbiology

3. Standard metric units

4. Prokaryotic Cells Revision: What’s a prokaryote?
What are the key features of prokaryotes?

5. Capsule
Cell wall
Ribosomes
Nucleoid
Flagella
Pili / fimbriae
Cytoplasm

6. Cell wall
Thick outer covering that maintains the overall shape of the bacterial cell

7. Ribosomes cell part where proteins are made
Ribosomes give the cytoplasm of bacteria a granular appearance in electron micrographs

8. Nucleoid
a ring of DNA

9. Flagellum
a whip-like “tail” that some bacteria have. Used for locomotion

10. Fimbriae hollow hair-like structures made of protein
allows bacteria to attach to other cells or surfaces
Fimbria - singular
Fimbriae - plural

11. Cytoplasm
clear jelly-like material that makes up most of the cell

12. So why are they this shape?
Bacterial Shapes
So why are they this shape?

13. Functions of the bacterial cell wall
Maintain the shape of bacterium
Rigid - protecting cell from osmotic lysis
Exchange nutrients and chemicals
Antigenicity
Related to its pathogenesis

14. Gram’s stain
To differentiate between Gram positive and negative bacteria

19. Growing bacteria in the lab
Bacteria can be grown if given the right conditions, nutrients and water.
There are a number of physical factors that effect bacterial growth:
Temperature pH
Oxygen availability

20. Physical Factors for bacterial growth
Temperature
Psychrophiles grow optimally below 15°C
Thermophiles multiply best around 60°C
Hyperthermophiles are Archaea that grow optimally above 80°C
Mesophiles thrive at the medium temperature range of 10° to 45°C, including pathogens that thrive in the human body

21. Most bacteria are happiest at 37oC and wont grow much about 42oC Some can produce spores and survive at very high temperatures e.g. Bacillus Spores can resist sterilisation and can be used as an indicator

23. Oxygen obligate aerobes require oxygen to grow
Anaerobes do not or cannot use oxygen
Facultative anaerobes grow either with oxygen or in reduced oxygen environments

26. pH The majority of species grow optimally at neutral (~7.0) pH
Acidophiles are acid-tolerant prokaryotes
For example, those used to turn milk into buttermilk, sour cream, and yogurt

27. These are supplied in a nutrient media
Nutrients
These are supplied in a nutrient media
Carbon – usually in an organic form eg glucose
Nitrogen – organic or inorganic
Other growth factors – vitamins and minerals
The usual source of energy used is glucose

28. Factors affecting bacterial growth
All of these factors can affect bacterial growth:
Extend lag phase
Decrease exponential (log) phase
Premature stationary/death phase

29. Bacterial growth curve

30. The ‘perfect’ growth curve
Can be used to calculate growth rate
Can be used to calculate generation time
Need linear part of growth curve
Calculate growth rate first
Calculate generation time once you know the growth rate

31. Generation times Linear part Growth rate (k) = log10Xt – log10X0 T
 Generation time = 1/(k)

32. arithmetic
semi-log
Rate of growth
Choose two points on linear part of graph
Higher value is Xt
Lower value is X0
Measure time interval between them (T)
Log the Xt and X0 values and put into following formula: T
Gives gen/hr (k)
Calculate generation time: gen time = 1/k
Gives answer in hr per gen convert to min/gen by multiplying the answer by 60
arithmetic
semilog

33. Handling cultures in the lab

34. Aseptic technique
Avoid contamination!

35. Sterile equipment
Equipment and media must be sterilised
Autoclave

37. Sterile equipment Equipment and media must be sterilised Autoclave
121˚C for 15mins
Under pressure
Bunsen for inoculating loops
Heat labile plastics are irradiated
Must be protected from contamination after sterilisation

38. Methods for measurement of cell numbers
Direct microscopic counts are possible using special slides known as a haemocytometer.
Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity.

40. Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface.
The sample or cell suspension can be diluted in a nontoxic diluent (e.g. water or saline) before plating. 
If plated on a suitable medium, each viable unit grows and forms a colony.
Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample.

41. Estimating size of viable bacterial population
4.5 ml
10-1
10-2
10-3
10-4
10-5
10-6
10-7
10-8

42. 1 ml
10-4
10-5
10-6
10-7
10-8
Gently swirl plate to mix

44. Make sure you work close to the bunsen burner
Pouring an agar plate
Make sure you work close to the bunsen burner
Flame the neck of the bottle

45. Motile bacteria Some bacteria are motile:
These tend to be Gram negative rods
Flagellar make bacteria motile
These can be stained
Unstained bacteria can be visualised swimming using light microscopy

46. Motility testing The hanging drop method:
Bacteria are suspended in a drop of liquid
They can be seen by light microscopy
Motile bacteria swim in straight line
Non-motile bacteria ‘vibrate’ a bit (Brownian motion)

47. Motile bacteria
Non-motile bacteria (Brownian motion)

48. Using bacteria commercially
A lot of commercial activities use microbiology directly in their processes
Food manufacturing (think yogurt, cheese, tofu and brewing)
Drug production
Waster water treatment, bioremediation etc
It is truly applied biology!

49. Streak plate to obtain a pure culture
Many processes require pure cultures to start with.
How do you do that in the lab? Streak Plates!

50. The microorganisms are grown in very large vessels called fermenters

52. The large stainless steel cavity is filled with a sterile nutrient solution, which is then inoculated with a pure culture of the carefully selected fungus or bacterium.
Paddles rotate the mixture so that the suspension is mixed well.
As the nutrients are used up, more can be added.
Probes monitor the mixture and changes in pH, oxygen concentration and temperature are all computer controlled.

53. A water jacket surrounding the fermenter contains fast flowing cold water to cool the fermenter since fermentation is a heat generating process.
Most of the air, including carbon dioxide and other gases produced by cell metabolism, leave the fermenter by an exhaust pipe.
There are two main types of culture used in industrial processes: batch cultures and continuous cultures.

54. Batch Culture
Continuous Culture
cells are grown in a fixed volume of liquid medium in a closed vessel
nutrients are added and cells harvested at a constant rate
No microorganisms, fluid or nutrients are added or removed from the culture during the incubation period
Volume of suspension is kept constant
Used for producing secondary metabolites, such as penicillin and other antibiotics, which are relatively unstable and not essential for the growth of the culture
Fermenter does not have to be emptied, cleaned and refilled very often
Secondary metabolites can be extracted economically only when they reach a high concentration in the culture
Production is almost continuous
Continuous cultivation needs sophisticated equipment to maintain constant conditions.  Highly trained staff need to operate the equipment.  Therefore this process can be expensive

55. Penicillin Production
Penicillium notatum (fungus) grown in batch culture
It produces the antibiotic after the growth (log) phase when glucose is depleted (limiting)
Can you think why it does that?
Made to reduce competition for its food
The fungal mycelium by filtration
The residual liquid is then processed to purify the antibiotic.

56. For more info on penicillin production: http://penicillin. wikispaces