Cultivation and control of microorganisms

Aseptic technique, microbiological culture media, selective and differential media, microbial growth. Laboratory safety; physical and chemical methods of control. Hazard groupings of microorganisms, containment categories for laboratories.

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Definitions for Sterilisation and Disinfection

  • Sterilisation= all microbes are removed including spores
  • Disinfection= killing, inhibition or removal of microbes that cause infections from inanimate objects (usually also results in reduction in the total microbial load)
  • Sanitisation= reduction of microbial load to a level considered safe.
  • Antiseptics= similar to disinfectants but used on living matter
  • 'Cidal and 'Static- e.g. virucide, fungicide, bacteriocide, bacteriostatic, fungistatic.

Hierarchy of resistance to killing

Spores High Resistance


Vegetative bacteria, small viruses, funghi

Lipid and larger viruses Low Resistance

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Physical Methods of Control

  • Moist heat
    • Boiling
    • Autoclaving Destruction
    • Tyndallisation
    • Pasteuristaion
  • Dry heat Destruction
    • Hot air oven
    • Direct flame
    • Incineration
  • Irradiation
    • UV Destruction
    • Ionising
  • Filtration
    • Depth filters
    • Membrance Filters  Removal
    • Air filters (safety)
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The Autoclave- Monitoring Methods

  • Spore strips
    • The most reliable method but also takes several days to get a negative result
  • Autoclave tape
    • Easy, convenient, inexpensive, but not as reliable
  • Browne's tubes
    • easy and inexpensive
  • Charts and printouts
    • built into most modern autoclaves
    • a thermometer can be used in ovens and steamers
    • must be calibrated or quality assured regularly
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Chemical Methods of Control

  • Phenolic compounds e.g. chloroxylenol (dettol)
  • Halogen-releasing agents e.g. sodium hypochlorite (bleach)
  • alcohols e.g. ethanol
  • quaternary ammonium compounds e.g. benzalkonium chloride
  • aldehydes e.g.formaldehyde
  • oxididing agents e.g. hydrogen peroxide
  • sterilising gases e.g.ethylene oxide
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Aseptic Technique- Why use it?

  • Microbes are everywhere
  • In the diagnostic laboratory, you must be sure that the microbes you grow really do come from the sample, not from the environment.
  • In hospitals and pharmaceutical industry, it is essential that drugs and equipment are microbiologically safe.
  • Taking samples from the environment, you need to avoid contamination from your own microbiota

So the aim of aseptic technique is to prevent contamination of sterile media or equipment during use


To prevent the contamination of pure cultures with unwanted environmental microbes.

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Key advice on aseptic technique

It takes practice!

Bunsen burner on a blue flame

Work as quickly as possible

Don’t put lids down on the bench if possible

Minimise air circulation

Don’t let non-sterile parts of pipettes come into contact with the inside of the “bottle”

If you think you have contaminated the end of a pipette then change it

Always dispose of pipettes/ pipette tips in the disinfectant provided – don’t put them down on the bench!

Use a laminar flow hood, if appropriate

Use a class II cabinet, if appropriate

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Bacterial growth- Binary Fission

Most prokaryotes reproduce by binary fission

  • Bacterial cell elongates
  • Chromosome is replicated
  • Two chromosomes seperate
  • Septum forms in mid-cell region
  • Parent cell divides into two identical daughter cells
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Measurement of microbial growth

1. Total Cell count (direct counting)

  • Direct microscopic count
  • Liquid samples
  • Use counting chamber

2.Viable counts

  • enumeration of living cells only
  • determines number of cells in a sample capable of forming colonies on a suitable agar medium
  • spread plate, pour plate, Miles and Misra, spiral plater

3. Measurement of culture turbidity

  • use a spectrophotometer- gives absorbance measurement- units- optical density (OD) for a particular wavelength (e.g. 540nm:OD540)
  • Plot OD data over time
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Measurement of Microbial Growth (2)- Other methods

  • Dry weight measurement
  • Automated methods - coulter counter, flow cytometry
  • Quatification of protein or DNA
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Phases of growth curve- Lag phase

Microbial cells inoculated into fresh culture medium:

  • lag before growth, due to cells adapting to new conditions


  • May be a change in nutrient medium
  • cells may be old, depleted of ATP, ribosomes
  • State of the inoculant: injured? spore form?
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Phases of growth curve- Exponential phase (log pha

  • Cells growing and dividing at maximal rate possible given their genetic potential and conditions under which they're growing.
  • Rate of growth is constant; cells are dividing and doubling in number at regular intervals
  • Population is most uniform in terms of chemical and physiological properties during this phase
  • Each microorganism is dividing at constant intervals (i.e. growth rate is constant)
  • Population will double in number during a specific length of time- the mean generation time or doubling time.
  • There is a direct relationship between the number of cells present in a culture initially and the number present after a period of exponential growth:
    • N0= initial population number
    • Nt=population at time t
    • n=Number of generations in time t
    • Nt = N0 x 2^n
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Phases of the growth curve- Stationary phase

  • Population growth ceased
  • total number of viable cells remains the same

Reasons for entering the stationary phase:

  • Nutrient limitation
  • O2 availability
  • Toxic waste products may accumulate
  • Several of these factors may act in concert.
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Phases of the growth curve- Decline (or death) Pha

  • Decline in cell number

Reason for entering this phase

  • Nutrient deprivation, toxic wastes.
  • Death of population usually logarithmic
    • death rate may decrease after population drastically reduced- some resistant cells may survive
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