Growth of Bacteria

Question 1

Describe how media can be used to cultivate and isolate bacteria

Answer 1

Agar plates are used with various types of media

Minimal media
Nutrient media
Enriched media
selective media
indicator medium
Selective indicator media
Transport media

Question 2

Describe minimal media

Answer 2

A basic salts based growth media often with a single carbon source.

Question 3

Describe nutrient Media

Answer 3

A medium with all the basic (general) requirements for growth but without specific supplements. (Often meat/yeast extract so a complex mix of peptides, ions and biochemical intermediates & salt)

Question 4

Describe enriched media

Answer 4

Nutrient media with additional organism specific supplements.

E.g. Selenite medium to enrich for Salmonella

Question 5

Describe selective media

Answer 5

A nutrient medium with the basic requirements for growth and added supplements that allows one bacterial type to grow more than another.

E.g. Specific antibiotics that allow isolation of slow growing bacteria

Question 6

Describe indicator medium

Answer 6

A medium with indicators which react to specific bacteria.

E.g. Lactose fermentation: E.coli (Lac+) & Salmonella (Lac-).

Question 7

Describe selective indicator media

Answer 7

A combination of selective and indicator media

E.g. MacConkey agar (s: bile salts, i: Lactose ferment)

Question 8

Describe transport medium

Answer 8

A medium designed to protect the organisms in you sample on the way to the lab.

E.g. swabs

Question 9

How to spread bacteria onto agar?

Answer 9

Spread bacteria on a plate until they are physically separated.
They then grow up to form individual colonies.

Each colony you see on a plate has grown from a individual bacteria.
If you have a mix of bacteria and spread them out you can separate the different bacteria.

Question 10

How does bacteria grow?

Answer 10

Grow by Binary fission

Colonies form on plates as piles of bacteria

Motile bacteria don’t tend to form colonies as they move quickly & spread out thinly

Bacteria have Generation times aka doubling times
= length of time required for a single bacterial cell to divide into two daughter cells

If bacteria double every 20-30min then colonies arise in 12-24 hours

Some slower bacteria can take months

Question 11

Once the bacteria has been culture and colonies formed, what happens next?

Answer 11

Tests can be done on the bacterial isolates:

Speciation through
	Molecular tests
	Biochemical tests

Antibiotic sensitivity

Typing (various methods) to determine epidemiology of infections

Question 12

How can bacteria be preserved for long term epidemiology?

Answer 12

Sub culturing (labour intensive & allows bacteria to change).

Freeze drying (lyophilisation).

Freezing with cryoprotection (DMS or Glycerol)

Question 13

Describe different methods by which you can measure bacterial growth

Answer 13

Direct counting by microscope.
You cannot distinguish viable from non-viable cells.

Colony counting (viable counts, below all basically variants of this).
Serial dilutions spread on surface of plate.
Mix bacteria with agar & pour plate.
Miles-Misra method.
Serial dilution steps
Membrane filtration.

Absorbance in liquid culture.
Measurement of absorbance of liquid culture at 600nm.

Question 14

Outline the Miles Misra Method

Answer 14

Diluted in log steps

Set volume is plated

The lower the dilution the fewer the amount of bacteria and so when the liquid dries the bacteria form separate colonies

The colonies from the 50µl (5 x 10µl) of the 10-4 dilution are counted

Using this it is then calculated up to how many would be in 1 ml.

The number are expressed using colony forming units / ml (CFU/ml)
These are a viable count for the bacteria in the original sample

Question 15

Outline the absorbance in liquid culture method

Answer 15

Increase in optical density over time.

Generation (time for one full round of division)

Growth rate (µ) how much doubling per hour.

Can measure by optical density

Question 16

Describe the basic growth characteristics of bacteria

Answer 16

Lag Phase
Bacteria adjust to the new medium & begin to metabolise & grow
Sensitive

Exponential Phase
Growth rate is determines
Bacteria grow at their best in this phase
More resistant

Stationary Phase
Bacteria reach limit on resources within the media
Maximum population has been reached
(where growth rate is = death rate)
Bacteriostatic antibiotics wont be as effective here as they prevent growth from
occurring

Decline / Death Phase
Medium exhausted of components for bacteria growth
New bacteria cannot grow and death rate remains the same

Measurement of death rate
	Drop Logs used to represent impact
To be aware that 90% / 99% drop doesnt always mean there is no bacteria left
	E.g a 99.9% drop of  (109) means  (107) still remains

Question 17

Define the terms anaerobic, aerobic, facultative anaerobe and microaerophilic

Answer 17

anaerobic ‘cannot grow in presence of oxygen’
Aerobic ‘require oxygen to grow’
Facultative Anaerobe ‘bacteria that can grow with or without oxygen present’
Microaerophilic ‘microbes which require very little oxygen to grow’

Question 18

What would the clinical relevance in knowing whether bacteria can grow in the presence of oxygen be?

Answer 18

A deep wound will be colonised by different bacteria based on oxygen gradient.
This can affect your treatment options

Question 19

Describe the impact on growth of different pH ratings

Answer 19

Most standard media is buffered at pH7

During bacteria growth acidic metabolites may be produced
This can lead to growth inhibition over time

Most pathogens are neutrophils (grow within 2 pH units of pH7)

	The acidophiles & alkaliphiles found in extreme environments are not 
            pathogenic
	Due to environments existing before first higher organisms developed

Question 20

Describe the impact on growth of different temperatures

Answer 20

The bacteria that infect or colonise animals are mesophilic (27 degrees)

Bacteria adapted to some animals have slightly different optima.
For example birds average temperature is 42°C and Campylobacter which is commonly found in birds grows best at 42°C. It has co-evolved for this environment.

That said it can also grow OK at mammalian body temperatures and cause disease.
 flexibility within a range

Question 21

At what temperature should bacteria be stored at to slow bacteria growth?

Answer 21

Approx 8°C
Slows bacteria growth but doesnt stop it completely

	Allows pathogens to survive and replicate outside/between hosts

Question 22

Describe the impact on growth of different osmolarities

Answer 22

Bacteria membrane is semi permeable
Accumulate or lose solutes to balance the water potential

Bacteria prefer to maintain a slight positive pressure an inflow of water which is resisted by the cell wall.

Question 23

What is the clinical relevance of knowing the preferred osmolarity for bacteria?

Answer 23

High salts/sugars can be used to preserve foods

Hypertonic environments draw water out of bacteria
leading to changes in the concentrations of solutes in the cytoplasm

Affects a range of enzymes/cell functions and so the changes disrupt cell physiology.

Question 24

Describe the core nutritional requirements for bacteria

Question 25

Where does bacteria obtain its nutrients?

Answer 25

Acquire nutrition from the immediate environment.
That environment may be your patients wound, intestine, blood

Bacteria can be:
Fastidious  require specific supplements

	Non-fastidious   grow from basic chemicals.

Most veterinary relevant organisms pathogens are chemoheterotrophs 
They use organic chemical as sources of energy & carbon

Question 26

How does knowing where bacteria obtain its nutrients and what it consume clinically relevant?

Answer 26

Part of the host defence is to limit access to certain nutrients (eg iron)
 reduced bacterial growth

Bacteria can compete for compounds so one organism can potentially block colonisation/infection by another

Question 28

Where are carbon and hydrogen sourced and what are they primarily used for?

Answer 28

Organic compounds (for chemoheterotrophs)

In many of the organic compounds in the cell

Question 29

Where is nitrogen sourced and what is its primary use?

Answer 29

Ammonia, Inorganic nitrogen (nitrates etc), already in organic molecules already containing nitrogen

amino acids, nucleic acids nucleotides, and coenzymes

Question 30

Where is sulphur sourced and what is its primary use?

Answer 30

In organic phosphates and already in some organic molecules containing phosphorous

Amino acids (cysteine, methionine, glutathione), some coenzymes

Question 31

Where are potassium, magnesium & calcium sourced and what are their primary uses?

Answer 31

salts

Co-factor
Potassium is also an osmolite

Question 32

Where is iron sourced and what is its primary use?

Answer 32

salts

Co-factor and component of cytochromes, non-haem iron-proteins

Question 33

Where are trace elements sourced?

Answer 33

metal ions required by certain cells in such small amounts that it is difficult to detect (measure) them. Not required to add them to most culture media as nutrients as in sufficient levels in other constituents

Question 34

Outline methods to control and manage pathogens in the environment

Answer 34

Sterilisation:
Sterilisation refers to a process that eliminates (removes) or kills (deactivates) all forms of life (in this case bacteria)
Main methods: heat, chemical, irradiations.

Washing.
Will not sterilize unless you use something that kills the microorganisms. But can remove and reduce risk of contamination or spread of infection.

Disinfection.
Do not assume that all disinfectants kill all pathogens.
Disinfectants may not be compatible with all uses.
Common methods are listed on next few tables make sure you understand what the terms refer to

Question 35

How does disinfection work?

Answer 35

There are a range of different chemicals used to disinfect.

They damage in a general manner by reacting with different targets such as proteins, nucleic acids
Most effects are to denature cell components  can damage/affect animal/human cells.

But prions very tolerant as already denatured so not fully effective or need extreme compounds.

Some are washes can be used on patients,
i.e. Chlorohexidine

Others are caustic or toxic.
i.e. formaldehyde, sodium hypochlorite, so need great care.

Selection must account for a lot of factors (few examples below).
Non tainting for food industry

Effectiveness in environment they will be used in (including temp and contamination type).

Effectiveness against the organism you want to target.

Question 36

Otline the methods of preserving and limiting bacterial growth

Answer 36

Refrigeration 4°C
Reduces growth rate of many organisms

Freezing
Prevents microbial replication

Boiling
Inactivation of vegetative bacteria and fungi but not endospores

Pasteurization
Inactivation of vegetative bacteria and fungi but not endospores or prions

Autoclaving (heat under pressure to 120°C or more)
Inactivation of all pathogen groups including endospores but not fully
effective against prions.

Acidification
Inhibits many bacteria. Weak acids used in food preservation pickling.

Increased osmotic pressure
Inhibits bacteria multiplication.

Irradiation
Inactivation agents, except Prions.