how to cultivate organisms in a microbiology lab

1. Cultivating organisms
Dr. Margaret Lubwama
3/2/15

2. The significant problems we face cannot be
solved at the same level of thinking we were
at when we created them…
Albert Einstein

3. Introduction
• The process of growing microorganisms is
referred to as culturing, the organisms grown
within a confined area being designated as a
culture
• The development of methods for producing
pure cultures, cultures that contain one type
of organism, was a major advance in
microbiology

4. Introduction
Koch
• Microorganisms caused
diseases
• Essential to study
organisms in pure
cultures
• Place microorganisms
onto a solid surface

6. Introduction
• Microorganisms grown on a
solid medium
-stay in one place, depending
on the extent of their motility
-each cell will give rise to a
large cluster of cells
-each cluster of cells is called a
colony
-cells in a colony are all
descendents of one original
cell
-if a new culture is inoculated
(started) from a single isolated
colony it is a pure culture

7. Introduction
• Which solid medium to
use
-cut surface of a potato
-gelatin
-supplemented with
various nutrients
• Bacteria could degrade
these substances (starch
in the potato and protein
in the gelatin), destroying
the solid surface
• Problem of
contamination

9. Agar
Agar:
-solidifying agent
-mixture of complex
polysaccharides produced
by several types of algae
genus Geledium
genus Gracilaria
-two major families of
polysaccharide
agarose: solidifies on its
own
agaropectin: doesn’t solidify
on its own

10. Agar
• Agar has the property of being solid until it is
heated to a temperature greater than 90C. It
will stay liquid down to about 40C
• Despite its polysaccharide nature, relatively
few bacteria are able to break it down into its
component sugars

12. Petri dishes
• ? Contamination
• Julius Petri: special glass
dish to hold bacteriologic
media
• Nested glass plates with
raised edges
• Permitted cultures to be
stacked in a small place
and transported from one
place to another without
contamination

13. Culture media
• A culture medium is any substance or mixture
of substances used to grow one or more kinds
of microorganisms in the laboratory
• A culture medium must fulfill all the
nutritional needs of the organisms to be
grown on it
• Source of energy as well as various chemical
elements to synthesize the molecules of which
it is composed

14. Bacterial growth curves
• Lag phase: period during
which cells are adjusting
their metabolism to
prepare for a new cycle of
growth
• Exponential phase: cells
are growing and dividing
at the maximum rate
possible for that medium
and those incubation
conditions

15. Bacterial growth curve
• Stationary phase: the
rate of production of
new cells exactly equals
the rate of death of old
cells, and the total
number of cells remains
constant
• Exponential death
phase: number of viable
cells in the culture
undergoes a rapid drop

16. Environmental parameters
Temperature requirement:
• Affects cellular enzymes and lipids of the cell
• Thermophiles: grow preferentially at high
temperature
• Psychrophiles: grow only at low temperatures
• Mesophiles: grow at moderate temperatures
Homework : Find out the temperatures and give
at least two organisms that fall in the above
groups

17. Environmental parameters
ph and salt requirements
• Water tends to flow into a typical cell causing it
to swell and possibly burst
• A very concentrated medium of high ionic
strength tends to draw water out of a cell,
literary dehydrating the cell even if it is
surrounded by water
• Organisms which require large amounts of salt in
the medium are halophiles
• Examples include…. 

18. Environmental parameters
Oxygen requirements
• Obligate aerobes need oxygen because they cannot ferment or respire
anaerobically
• The generally accepted definition of an anaerobe is an organism that
requires reduced oxygen for growth. Practically, this means that an
anaerobe fails to grow on the surface of solid media in 10% CO2 in air
- Obligate anaerobes are poisoned by oxygen
• In contrast, facultative organisms can grow in the presence or absence of
air. Facultative anaerobes can grow with or without oxygen because they
can metabolise energy aerobically or anaerobically
• Microaerophilic: need oxygen because they cannot ferment or respire
anaerobically. However, they are poisoned by high concentrations of
oxygen

19. Definition
Broth containing
microorganisms
AIR

20. Environmental parameters
Nutrients
• Carbon
• Nitrogen
• Magnesium
• Potassium
• Iron
• Sodium

21. Media types
• Solid media
• Liquid media
• Semi solid media

22. Different types of media
• Basic media: simple media that will support
the growth of microorganisms that do not
have special nutritional requirements, e.g.,
nutrient agar, nutrient broth

23. Different types of media
• Enriched media: these are prepared to meet the nutritional
requirements of more exacting bacteria. It supports growth of
pathogens that require additional nutrients or growth
stimulants. By addition of blood, serum, egg… e.g., blood
agar, chocolate agar
• Enrichment media: used to describe a fluid medium that
increases the numbers of pathogens by containing
enrichment that selectively favor it or inhibitory substances
that suppress competitors, e.g., selenite F for Salmonellae

24. Different types of media
• Selective media: one that allows only a certain organism or
group of organisms to grow while preventing growth of all
other organisms
MacConkey agar selects for Gram-negative organisms. Has
inhibitors for Gram-positive organisms- Bile salts, Crystal
violet
• Differential media: colonies growing on them display some
sort of readily visible reaction indicative of one aspect of their
metabolism
MacConkey agar differentiates between lactose fermenters
and non-lactose fermenters

25. Streaking

26. Interpreting media
• Describe the colonies
• Describe effects of colonies on the different
media used
• Performing an initial Gram stain is important.
Why do you think so?

27. Characteristics of colonies
• Size: diameter in millimeters
• Elevation: flat, raised
• Margin (edge of colony): entire, curled
• Color: white, yellow, black, buff
• Surface: glistening, dull
• Consistency: mucoid

28. Reaction in agar media
• Hemolysis on blood agar
• Pigment production in agar medium

29. Bacteria families that can grow in
conventional labs
• Micrococcaceae
• Streptococcaceae
• Enterobacteriaceae
• Pseudomonadaceae
• Campylobacteraceae
• Vibrionaceae
• Neisseriaceae

30. Bacteria that require special
conditions
• Mycobacteriaceae
• Spirochaetaceae
• Brucella spp
• Mycoplasma spp
• Chlamydia spp

31. T.B media
Lowenstein Jensen media for Mycobacterium spp.

32. Incubator Loops
Petri Dishes