Pure Culture Techniques

(Lab Exercise 9)
 Culturing Microorganisms
Microorganisms are ideal lab subjects
- grow rapidly
-are small in size
-many generations can be
observed
-results can be applied to other
organisms
 Pure Culture
Consists of only a single type of
organism.
Rarely exist in nature. Naturally
exist as mixed cultures.
Pure cultures must be obtained
artificially
Pure Culture techniques

Goal: To isolate a single species of bacteria form

a mixed population.
 Isolation
• Microorganisms occur in huge numbers.
• *Isolation of single species (pure culture)
is done by :
• Streak plate method
• Pour plate method

*Based on diluting sample out to a point where

a single cell will give rise to a single colony
Streak Plate
(Quadrant Streak)
Pour Plate
* We will do loop dilution
technique (see fig 9.5)
 Growing a Pure Culture
• To grow microorganisms:
– nutrients must be supplied in the
culture medium
(Agar is the solidifying material used in solid media. It is
an extract of seaweed that melts at 100 C and solidifies at
o

about 42 C)
o

– A suitable environment must be

provided
 Culture Medium
• Enriched Media- is formulated with all the
necessary ingredients for a wide variety of organisms
and grows a multitude of bacterial species (example:
Trypticase Soy Agar –TSA)
• Selective Media- are formulated with
ingredients that inhibit the growth of some bacteria,
such as an antibiotic, but enhance growth of the target
organism (example: Blood Agar –BA)
• Differential Media- includes ingredients,
such as chemical indicators, that produce observable
differences between species of bacteria. (example:
MacConkeys agar –MAC)
 Suitable Environment
• Temperature
• pH
• Oxygen
 Temperature
• Microbes grow over a range of
temperatures ( nat. environ)
• Temperatures maintained in
–Incubators
–Waterbaths
 pH
• Optimal pH varies
• Bacteria neutral pH 6.5-7.5
• Buffers are added to neutralize the
by-products of bacteria
metabolism
 Oxygen
• microbes respond differently
–Strict aerobes
–Strict anaerobes
–facultative aerobes
–facultative anaerobes
–Microaerophiles
O2 must be provided , limited or
excluded
Colony morphology
Colony morphology
Enumeration of Bacteria
(Lab Exercise 20)
 Methods for Determining Bacteria
Numbers
Goal: To estimate the number of bacteria in a sample

Possible Methods (We will not do these):

1)Microscopic Counts
- Uses special slides with a grid (Petroff-Hauser slides)
- Counts dead and living cells
- Must know exact volume
2) Most Probable Number Counts
- Used in large scale applications
- tracks metabolic by-products (e.g. acid/gas in coliform
bacteria) and compares results to established statistical
tables to estimate probable numbers of bacteria in sample
- Counts Dead and living
Microscopic Counts
(Petroff-Hauser slides)
 Methods for Determining Bacteria
Numbers continued
3) Standard Plate Count
- Sample serially diluted and
dilutions plated
- Count multiplied by dilution
factor
- Technique assumes that 1 cell
will give rise to 1 colony
(colony forming unit (CFU)
- Counts ONLY living (viable)
organisms!

(We will do this method)

 Standard Plate Count

Chapter 6
Chapter 6
 Determination of Growth by Optical
Density
• Turbidity (cloudiness)
– Measured by a spectrophotometer at wavelength 686nm
– Scale
• %Transmittance (amount of light passing through)(measured)
• Optical Density or Absorbance(calculated)

To convert between the optical density (OD)and transmittance(T)

scales, use the equation:

OD = 2 – Log (%T)

Chapter 6
Chapter 6
Chapter 6