Cultivation and Isolation of Microbes

Objectives
Students should be able to
 Define microbial culture

 Define pure culture

 Describe the purposes to culture microorganisms

 Demonstrate and explain the use of aseptic techniques,

culture media, and controlled environments for culturing
and isolating microorganisms
 Explain batch and continuous cultures

1
Cultivation and Isolation of Microbes

Why Grow Microorganisms?

 To study their characteristics and behaviour:
morphology, growth requirements, genetics, etc
 To produce food, commodity chemicals, pharmaceuticals
and other useful products
 To diagnose infectious diseases by detecting and
identifying pathogens
 To detect microbial contamination in environment, food,
etc

2
Cultivation and Isolation of Microbes

 A culture – a collection of microorganisms

 To culture – to make a culture
 To subculture – to make a culture from a pre-existing
culture

3
Cultivation and Isolation of Microbes

Inoculum

Incubate

A pre-existing Fresh medium A new culture

culture

4
Cultivation and Isolation of Microbes

 To make a culture, a sample called the inoculum (pl.

inocula) is introduced into a suitable medium (a collection
of nutrients; pl. media); the act of doing so is called
inoculation (v. inoculate)
 The inoculum can be taken from any source (soil, water,
etc) or from another culture
 The inoculum together with the medium is placed in a
controlled environment (in an incubator) to encourage
the microorganism(s) to grow; the act of doing so is
called incubation (v. incubate)

5
Cultivation and Isolation of Microbes

Culture Media
 Two types: liquid (broth) and solid (agar)

 Choice depends on purpose of culture and type of

microbe
 Some microbes can be cultured only on one type of
medium, eg. many algae, most free-living protozoa, and
some bacteria can only be cultured in broth

6
Cultivation and Isolation of Microbes

Mixed and Pure Cultures

 Mixed culture – a culture that contains more than one
species of microbes
 Pure culture – a culture that contains a single species of
microorganism
Mixed culture Pure culture
(Many different types of colonies) (Single type of colonies)

Agar

7
Cultivation and Isolation of Microbes

Broth Culture
 Growth appears as turbidity

 Large quantities of medium can be Non-inoculated

prepared easily broth is clear

 Large quantities of microbial cells can

be cultured
 Easily harvested and diluted

 Difficult to tell whether it’s contaminated,

and impossible to salvage if Microbial growth in
contaminated broth causes it to
become turbid

8
Cultivation and Isolation of Microbes

Solid Culture Colony

(comprises
 Microorganism grow as colonies on the millions of cells)
surface of the agar (each colony arises
from a single cell)
 Useful for isolation to give pure cultures

 Easier to detect for contamination

 More tedious to prepare, esp in large

quantities
 Smaller yields and more difficult to
harvest

9
Cultivation and Isolation of Microbes

What Do You Need to Grow Microorganisms?

 Aseptic techniques

 Sterilized media and Equipment

 Knowledge of Growth Requirements

 Controlled Environment

10
Cultivation and Isolation of Microbes

Aseptic Techniques
 A set of specific practices and procedures performed
under carefully controlled conditions so that there is
minimal contamination by undesired organisms
 To protect samples, cultures, sterilized media and
equipment from contamination by surrounding
microorganisms in air, hands, work bench, etc
 To protect user from potentially harmful microorganisms

11
Cultivation and Isolation of Microbes

Sterilized Media, Materials and Equipment

 Media, materials and equipment need to be sterilized to
destroy microorganisms in/on them before use
 To ensure only intended microorganism is cultured

 In the lab, sterilization can be achieved through

 Autoclaving

 Heating in hot air oven

 Flame sterilization

 Filtration

12
Cultivation and Isolation of Microbes

Autoclaving
 Subject media, materials and equipment to steam under
pressure – steam sterilization
 Must be thermostable and not sensitive to moisture

 Kills vegetative cells and endospores

 Most common settings

 121oC, 15 psi, 15 min

o
 115 C, 10 psi, 10 min

psi: pounds per square inch

13
Cultivation and Isolation of Microbes

Heating in Hot Air Oven

 For materials and equipment that may be sensitive to
moisture
 Up to 160-180oC for several hours

 Kills vegetative cells and endospores (depends on

temperature and duration)

14
Cultivation and Isolation of Microbes

Flame Sterilization
 For non-flammable equipment that are used over and
over again within a short time frame
 Eg.

 Inoculating loop and wire (flame till red hot)

 Glass spreader, metal scissors, metal spatula (surface

sterilization using alcohol and flame)

15
Cultivation and Isolation of Microbes

Filtration
 For fluids and solutions that are not thermostable

 Pass fluid/solution across filter membrane

 Based on size exclusion; microbial cells are trapped on

membrane because they are too large to pass through
the pores of the membrane
 Therefore filtrate is sterile

16
Cultivation and Isolation of Microbes

Filtration (cont’d)
 Common membrane pore sizes:

 0.45mm – good for removing bacterial and larger cells

 0.22mm – good for removing large viruses

17
Cultivation and Isolation of Microbes

Controlled Environment
 To provide optimal environmental conditions for
microbial growth
 Incubators with temperature and/or humidity control are
used to provide the optimal conditions to encourage
microbial growth

18
Cultivation and Isolation of Microbes

Controlling pH and Osmotic Pressure

 Medium is carefully formulated to be a roughly isotonic

 pH is also adjusted such that the medium supports

optimal growth of the particular microbe
 pH is maintained by using buffers (combination of a
weak acid and its salt)

19
Cultivation and Isolation of Microbes

Controlling Gaseous Atmosphere

 Obligate aerobes and facultative anaerobes – no extra
requirements
 Obligate anaerobes – anaerobic gas jar, anaerobic
chamber
 Microaerophiles and capnophiles – candle jar, CO2
incubators

20
Cultivation and Isolation of Microbes

Isolation
 The act of getting a pure culture from a sample and/or
mixed culture
1. Using solid media, attempt to distribute or separate the
cells such that each cell grows into an individual colony
which can be picked up
 Streak plate method
 Pour and spread plate methods
2. Using a series of tubes, dilute or sequentially inoculate
the sample until a single cell is present – grows into pure
culture

21
Cultivation and Isolation of Microbes

2
Streak Plate Method
3

= Flame-sterilised the inoculating

loop until red-hot and cool down
before the next streak

22
Cultivation and Isolation of Microbes
Serial dilution, pour and spread plate methods

Culture

23