Lecture 2 - Bacterial nutrition

Question 1

Culture media

Answer 1

a solid or liquid preparation use to grow, transport and store micro-organisms. To be effective the medium must contain all the nutrients the microorganism requires for growth

Question 2

Defined media

Answer 2

Chemically defined, know the exact composition, know every component and the amount of every component as every component is added individually

Made with specific chemicals - specific sugars, amino acids, vitamins

Less common

Grows most chemoautotrophs and photoautotrophs (photoautotrophs use CO2 as a carbon source and light as an energy source therefore they can be grown on media containing sodium carbonate or bicarbonate (sources of CO2), nitrate or ammonia as a nitrogen source, sulphate, phosphate and other minerals)

Question 3

Complex media

Answer 3

Do not not the exact composition, not chemically pure

Use complex sources of nutrients - glucose (source of energy and carbon), beef extract, yeast extract (both the beef and yeast extract supply water soluble acids that are rich in nitrogen and carbon), peptones ( digestive protein material)

Most common

Grows most chemoheterotrophic organsims

Question 4

Transport media

Answer 4

Should maintain the viability of all organisms in the specimen without cell division - Want specimen to arrive at the lab in exactly the same condition as it was taken from the patient

Contains only buffers and salts (there for protective purposes)

Lacks carbon, nitrogen and organic growth factors (since you do not want any microbial growth happening)

Question 5

The two words to describe transport media….

Answer 5

temporary storage

Question 6

Enriched media

Answer 6

Contains general nutrient supplements (e.g. serum or yeast extract), to provide precursor metabolites

Made to accommodate lots of bacteria even the fastidious (fussy) ones

Would use when you are doing a bit of a survey about what microbes are present

Nutrient supplements to help grow a wide range of bacteria

Good for when you do not know what you are growing

Question 7

Enriched media two words

Answer 7

generally encouraging

Question 8

Enrichment broths

Answer 8

Encourage the growth of one particular organism, put things in it that will get a specific microbe to grow

Gives a competitive edge to the desired microbe, which then becomes the dominant species ( not hidden anymore by the other microbes)

Commonly used in the clinical laboratory - to culture human samples since humans have lots of microbes and the ones we are investigating are often pathogens which can be present in low numbers

Question 9

Enrichment broth example

Answer 9

Salmonella

Low concentration of salmonella in faecal specimen so an enrichment broth is used to promote salmonella growth so that it can be detected

Tetrathionate broth holds the normal flora in the lag phase of growth whilst promoting logarithmic growth of salmonella as it has the tetrathionate reductase enzyme

Concentration of pathogens is usually low, concentration of intestinal normal microflora is high

Question 10

Enrichment broth two words

Answer 10

Specifically encouraging

Question 11

Selective media

Answer 11

Encourages the growth of some organisms while specifically inhibiting the growth of others
Used in clinical samples

Question 12

Selective media 2 words

Answer 12

Specifically inhibiting

Question 13

Mannitol salt agar

Answer 13

Differential and selective

Selective
7.5% sodium chloride - inhibits all except staphylococci therefore it is selective so staphylococci
Selective ingredient which is 7.5% sodium chloride + general nutrients to support microbial growth
Inhibits all gram negative, inhibits most gram positive excluding staphylococci (gram positive)

Differential 
Mannitol is a carbohydrate so microbes that can ferment will react with this and produce acid which results in a pH change shown by the phenol red 
Phenol red (pH indicator) 
Mannitol fermented = acid 
Mannitol fermenters (S. Aureus) change the agar colour from pink to yellow = pathogens 
Non-mannitol fermenters (S.epidermidis) agar colour not changed (remains pink) 
S. Aureus (pure culture) - can tolerate the selective ingredient and so it grows and it also is a mannitol fermenter so the pH indicator picks up on this and there is a colour change from pink to yellow 
S. Epidermis - can grow since it can tolerate the selective ingredient which is the 7.5% sodium chloride but it cannot ferment the mannitol which is the differential ingredient therefore it remains pink in colour

Question 14

Hektoen agar

Answer 14

Differential and selective, has 7 different ingredients

selective
Bile salts (selective ingredient) + general nutrients
Inhibits gram-positive and some (non-enteric) gram negative bacteria
Selective for gram-negative enteric (gut) bacteria

Differential
3 carbohydrates = lactose, sucrose and salicin
2 pH indicators = acid fuchsin, bromothymol blue - which allow us to visualise if any carbohydrates are being utilised
Carbohydrate (s) fermented => acid (yellow/pink)
Fermenters e.g. E. coli will turn the agar yellow/pink
Nonfermenters e.g. shigella and salmonella (pathogens) colony colour is blue/green
Sodium thiosulphate and ferric ammonium citrate work together
FAC is a source of iron which allows the production of hydrogen sulphate from sodium thiosulphate, H2S production gives black colonies or black centres
H2S creates black colonies int he middle of salmonella (blue/green with black centres) but not shigella (blue/green) therefore can be differentiated from other members of the enterobacteriaceae family (yellow/pink)

Question 15

Differential media

Answer 15

Contains indicators that visually distinguish between organisms - usually a colour change
Can give a preliminary or preemptive result

e.g. mannitol salt agar and Hektoen agar

Question 16

Differential media two words

Answer 16

visually distinguishing

Question 17

Blood agar

Answer 17

Enriched = blood 
Differential = blood 
Bacterial enzymes (haemolysins) act on the animal cytoplasmic membrane (red blood cell membrane and it forms a pore that results in the lysis of the RBC) 
Observe by streaking organism onto blood agar, once bacterial growth has begun the haemolysin is release and lyses the surrounding the red blood cells, creating a zone of haemolysis 
Types of haemolysis 
Alpha = partial lysis of RBC —> green discolouration 
Beta = complete lysis of RBC —> complete clear zone 
Gamma = non-haemolytic

Question 18

Blood agar two words

Answer 18

enriched and differential

Question 19

Batch culture

Answer 19

Closed system
Nothing is added or removed during the growth of a culture
Displays the growth curve

Question 20

Continuous culture

Answer 20

Not a closed system, continuously adding nutrients in and removing waste
Known as chemostats
Similar to the human digestive system
Want to keep bacteria in the exponential phase because they may produce components that are useful to us
Rate of growth determined by the rate of addition of fresh medium into the culture flask

Question 21

Turbidostat

Answer 21

Second type of continuous culture system and it has a photo cell that measures the turbidity (defined as the amount of light scattered) of the culture in the growth vessel

Question 22

Chemostat

Answer 22

Most simple of the continuous batch culture devices

Constructed so that the rate at which a sterile medium is fed into the culture vessel is the same as the rate at which the medium containing microorganisms is removed

Question 23

Primary metabolite

Answer 23

Essential for growth of the microbe
Produced during the logarithmic/exponential phase
Mirrors growth because the primary metabolites are formed in the exponential phase of growth
Such as amino acids, nucleotides and fermentation products such as ethanol and organic acids

Question 24

Secondary metabolite

Answer 24

Not essential for growth e.g. antibiotics - But very useful if you are antibiotic producing microbe because you have a competitive advantage and can kill off other microbes that might be in the environment around you
Accumulate during stationary phase (accumulate when nutrients become limited or waste products accumulate following the active growth phase) - Most antibiotics fall into this category - If we wanted to harvest these we would set up a chemostat differently so that it would keep the bacteria in the stationary phase

Question 25

Four phases of the microbial growth

Answer 25

Lag
log/exponential
stationary
death

Question 26

what has the characteristic growth curve?

Answer 26

The theoretical growth of a bacterial batch culture (liquid), incubated under ideal conditions has a characteristic growth curve

Question 27

Lag phase

Answer 27

The time required to get biosynthetic reactions running, lag between adding the cells to the media to when it actually starts growing

It not a time of inactivity but it is rather cells are synthesising new component which are needed before growth can begin

Question 28

Log/exponential phase

Answer 28

Cells are actively dividing and nothing is limiting for growth

During this phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium and the environmental conditions

Population is doubling in a constant time interval which is determined by the mean generational time

Slope reflects the rate of division … steep = short mean generation time i.e. the numbers build up quickly

Question 29

Stationary phase

Answer 29

Total number of surviving cells remains constant

Numbers are static - cells still forming by binary fission are mirrored by cell death

Nutrients used up (nutrient limitation) or waste becomes toxic is what causes this phase

Question 30

Death phase

Answer 30

Exponentially decreasing, cell death is outnumbering cell growth, cells dying at a constant rate

Death = unable to reproduce

Question 31

Long-term stationary phase

Answer 31

long-term stationary phase is a highly dynamic period in which the ‘birth’ and ‘death’ rates are balanced. That is, long-term batch cultures have an apparent carrying capacity that can only support a certain number of cells.

Long-term growth experiments reveal that after a period of exponential death some microbes have a long period where the population size remains more or less constant

Question 32

How to calculate the no. of bacteria during exponential growth

Answer 32

Final number = initial number x 2^n where n= generation number and initial=no. of microbes before growth began

Question 33

Direct count

Answer 33

Advantage = quick 
Disadvantage = counts both live and dead cells 
Haemostatometer = physically count the cells 
Spectopertrometer = counts cells directly but through the optical density (turbidity)

Question 34

Viable count

Answer 34

Advantage = only counts live cells
Disadvantage = slow
Dilute culture - plate culture - one cell = one colony on plate which we then count

Question 35

Gram positive

Answer 35

thick layer of peptidoglycan in bacterial cell wall which traps crystal violet masking the red dye

Question 36

Gram negative

Answer 36

thin layer of peptidoglycan with inner and outer membranes which means that crystal violet is easily rinsed away, revealing the red dye

Question 37

Organisms most likely to survive extreme

Answer 37

Archaea