Media

Question 1

Pure culture

Answer 1

population of cells arising from a single cell

Question 2

who developed pure culture isolation

how?

Answer 2

Koch

• Earlier used potato slices, then gelatin
  Many organisms can digest gelatin and it melts at 37°C
• Later used agar (from seaweed)
  Revolutionised microbiology

Question 3

Advantages of using agar as media

Answer 3

Melts at 90-96°C, sets at about 45°C

• Rarely digested by bacterial enzymes
• Can incubate at higher temperatures, without agar melting
• Can add antibiotics, blood etc to molten agar at 46-50°C

Question 4

Plating methods

Answer 4

Streak plates - Spread a culture to isolate separate cells
Ensure purity or to separate mixtures

• Diluted by streaking with a flamed loop
• Individual cells, each grow into one isolated colony

Question 5

Streak plates method

Answer 5

Collect sample on sterile loop
Smear onto part of plate
FLAME AND COOL LOOP
Streak 4 lines on plate
FLAME AND COOL LOOP
Streak 4 lines on plate
FLAME AND COOL LOOP
Streak 4 lines on plate
Use up rest of plate with streak

Only touch first streak once

Question 6

Requirements for laboratory culture

Answer 6

• Solid or liquid medium
  = Must supply all the nutrients a microbe needs
• Different microbes have different needs
  = Knowledge of normal habitat can help

-Specialised media grows specific microbes
= Isolation of clinical pathogens, or in food, water
= Hydrocarbon utilisation
= Growth low pH

Question 7

Defined media

Answer 7

• chemically defined
• concentration of each constituent known
• specific source of CHNOPS + ions

Question 8

Support growth media for BG-11 - cyanobacteria

Answer 8

Added CO3^2- for photosynthesis

• it is a photoautotroph, CO3 provides CO2 for photosynthesis
• Identify- CHNOPS, K, Ca, Mg and trace metals
• Final pH 7.4

Question 9

Support growth media for E. coli growth medium

Answer 9

Glucose minimal medium – controlled C-source

• As it is a chemoorganoheterotroph – using glucose as C and energy source; ammonium as N source
• require high levels of each component for growth, in particular P.
• No amino acids provided, as it synthesise own
• Final pH 7.0
• Change C- source/ omit Fe to study effect
• Test mutants for utilisation of Cho, synthesis of amino acid

Question 10

Support growth media for CDM - Haloarchaea

Answer 10

High salt concentration

Question 11

Uses of defined media

Answer 11

Selectivity

• Specific carbon sources
• Limits to other nutrients

Use selective media to “enrich” specific organisms
- effective isolation media eg bacteria capable of degrading oil or mutants unable to utilize a specific C-source

Also used to assay compounds by monitoring growth eg Vitamin assay

Question 12

To isolate bacteria that rapidly devour hydrocarbons in oil spill:

What would you put in the growth media?
To develop a spray using oil degrading- bacteria– what nutrients would you add to the spray?

Answer 12

Oil/ Hydrocarbon as sole carbon source. Plus source N, S, P etc.

If eventually used as spray need to ensure good supply of N,S and P for rapid growth

Question 13

Complex media

Answer 13

= Components of undefined composition (unknown)

• Include digested proteins (casein)
• Extracts of complex substances (Beef extract, Yeast extract)

= General purpose media supply needs of many different microbes
- Nutrient broth, trypticase soya broth

= Additives for enriched media

• blood to support growth of pathogens
• Low pH eg Malt agar pH 6.0, lactic acid bacteria, yeast

Question 14

Complex media is useful when

Answer 14

when growth requirements are not known or for optimal growth, show composition of MacConkeys

Question 15

Selective media

Answer 15

Favour growth of particular organisms, inhibit growth of others

• Specific substrates
• Inhibitors
• pH eg Malt agar

Question 16

Differential media

Answer 16

• Distinguish between organisms

- Can allow tentative identification

Question 17

MacConkey agar

Answer 17

a selective and differential medium
with casein / animal tissue / Lactose / Bile salts / NaCl / Neutral red / Crystal violet

Selection - Bile salts (inhibitor)
: selects for bacteria able to grow in the gut (coliforms)

Differentiation - Lactose + pH indicator : lactose fermenter –pink (eg Escherichia coli)
Non-lactose fermenter – white (eg Salmonella spp.)

Question 18

How to ckeck the quality/ safety of the source of drinking water?

Answer 18

Quantitate - the heterotrophic, aerobic colony count

Estimate faecal contamination

Question 19

How to estimate faecal contamination

Answer 19

using MacConkey broth (E. coli as an indicator) – acid and gas production following growth at 37C

Question 20

Anaerobic chamber

Answer 20

• Vacuum pump + N2 purges
• Palladium catalyst and hydrogen to remove remaining O2
• Interchange compartment to prevent exposure to O2

Question 21

Purity checks

Answer 21

Single colony and cell form
No extra growth on non-selective media
[same bacteria will give same colony and cell form under same growth conditions]

Question 22

Anaerobic jar

Answer 22

growth in absence of O2

O2 removed from chamber by combineing with H to form H2O, catalyzed by palladium pellets

Water is added to chemicals in envelope to generate H2 & CO2. Carbon dioxide promotes more rapid growth of microbes.

Methylene blue becomes colourless in absence of O2

Question 23

Solid growth medium

Answer 23

• Agar plates – growth medium mixed with solid support (agar)
• Used for general maintenance of cultures
• Used for separating mixed cultures
• Small scale growth of bacteria

Question 24

Growth in liquid culture

Answer 24

Batch culture

Continuous culture

Question 25

Batch culture

Answer 25

a closed system culture of microorganisms of finite volume with specific amounts of nutrients and environmental conditions. Only a few generations are possible before nutrients are depleted.

Question 26

Bacterial growth

Answer 26

Lag phase log phase/ exponential phase Stationary phase Death phase

Question 27

Lag Phase

Answer 27

When innoculated cells usually do not start to grow immediately Cells are synthesising new cellular material Innoculated cells often depeleted in energy and essential cellular components (e.g. ATP, ribosomes, enzyme cofactors) Need to prepare for growth

Question 28

Log (exponential) Phase

Answer 28

= Cells are growing at maximum rate For many this is exponential Culture is mostly chemically and physiologically uniform Ideal for biochemical/physiological studies = Balanced growth All cellular components in balance In up-shift or down-shift balance is disturbed Growth decreases until balance resumed

Question 29

Stationary phase

Answer 29

Possible causes: - Nutrient depletion - Build-up of toxic waste-products, e.g. Acid production during fermentation - Population reaches a maximal size (quorum sensing) Once growth stops: - Continued growth balanced by cell death or no growth but still metabolic activity

Question 30

Death phase

Answer 30

Number of viable cells slowly declines - Can be logarithmic - Dependent upon conditions Death rate can slow as numbers decrease - Survival of resistant cells

Question 31

Continuous culture

Answer 31

a culture of microorganisms in a medium which is maintained under constant environmental conditions with a constant nutrient supply so that it can grow over an extended period of time. Achieved in a chemostat -Steady state conditions.

Question 32

Maintenance of steady state conditions.

Answer 32

Media fed into the chemostat at a steady rate. Biomass and spent media removed from chemostat at the same steady rate. Bacterial growth phase maintained.

Question 33

Batch culture Advantages

Answer 33

Versatile: can be used for different reactions every day.  Safe: can be properly sterilized.          Little risk of infection or strain mutation. Complete conversion of substrate is possible

Question 34

Batch culture Disadvantages

Answer 34

High labour cost: skilled labour is required.  Much idle time: Sterilization, growth of inoculum, cleaning after the fermentation.  Safety problems: when filling, emptying, cleaning.

Question 35

Continuous culture Advantages

Answer 35

Works all the time: low labour cost, good utilization of reactor. Often efficient: due to the autocatalytic nature of microbial  reactions, the productivity can be high.  Automation may be very appealing. Constant product quality.

Question 36

Continuous culture disadvantages

Answer 36

Often disappointing: promised continuous production for months fails due to a. infection. b. spontaneous mutation of microorganisms to non producing strain.  Inflexible: can rarely be used for other productions without substantial modification.

Question 37

Measurement of Culture Density

Answer 37

Counting plate: Plate serial dilutions of known volumes and count number of colonies. Counting haemocytometer: a thick glass microscope slide with a known volume of culture placed on it. Measure optical density (must count first).