Cultivation of
Bacteria
Muhammad Hilal Jan
Lecturer MLT
Cultivation of Bacteria
Process of allowing microbes to grow on
artificial medium/media in order to
identify them and to observe their growth.
Inoculation of Bacteria
In Microbiology, inoculation refers to the
act of introducing Bacteria or suspensions
of bacteria to a culture Media
Inoculation Method depend on the
purpose for which they are intended
How to inoculate Culture
Media/Protocols to follow
– Immediately before inoculating a culture medium, check the
medium for visual contamination or any change in its appearance
which may indicate deterioration of the medium, e.g. darkening in
colour.
– When inoculating, or seeding, culture media an aseptic (sterile)
technique must be used. This will:
– Prevent contamination of cultures and specimens
– Prevent infection of the laboratory worker and the environment.
Aseptic Techniques
Flame sterilize wire loops, straight wires, and metal forceps before and after use
Flame the necks of specimen bottles, culture bottles, and tubes after removing and before
replacing caps, bungs, or plugs.
When inoculating, do not let the tops or caps of bottles and tubes touch an unsterile
surface. This can be avoided by holding the top or cap in the hand
Always use racks to hold tubes and bottles containing specimens or culture media.
Make slide preparations from specimens after inoculating the culture media.
Decontaminate the work bench before starting the day’s work and after finishing.
Use a safety cabinet when working with hazardous pathogens
Wear protective clothing, wash the hands after handling infected material, and never mouth
pipette, eat, drink, or smoke in the laboratory
Inoculation Methods
Streak Culture Lawn Culture Stroke Culture Stab Culture
Anaerobic
Pour Plate
Liquid Culture culture
Method
Methods
Streak Culture
Used for the isolation of bacteria in pure culture from clinical specimens.
Platinum wire or Nichrome wire is used.
One loopful of the specimen is transferred onto the surface of a well dried
plate.
Spread over a small area at the periphery.
The inoculum is then distributed thinly over the plate by streaking it with a
loop in a series of parallel lines in different segments of the plate.
On incubation, separated colonies are obtained over the last series of
streaks.
Lawn Culture
– Provides a uniform surface growth of the bacterium.
– Uses
– For bacteriophage typing
– Antibiotic sensitivity testing
– In the preparation of bacterial antigens and vaccines.
– Lawn cultures are prepared by flooding the surface of the plate
with a liquid suspension of the bacterium.
Stroke Culture
Stroke culture is made in tubes containing agar slope
/ slant.
Uses
• Provide a Pure growth of bacterium for slide agglutination and
other diagnostic tests.
Stab Culture
Prepared by puncturing a suitable medium – gelatin or glucose
agar with a long, straight, charged wire.
Uses
• Demonstration of gelatin liquefaction.
• Oxygen requirements of the bacterium under study
• Maintenance of stock cultures.
• Motility Test
Pour Plate Culture
Agar medium is melted (15 ml) and cooled to 45oC.
1 ml of the inoculum is added to the molten agar.
Mix well and pour to a sterile petri dish.
Allow it to set.
Incubate at 37oC, colonies will be distributed throughout the depth of the medium.
Uses
• Gives an estimate of the viable bacterial count in a suspension. – For the quantitative urine cultures.
Liquid Culture
Liquid cultures are inoculated by touching with a charged
loop or by adding the inoculum with pipettes or syringes.
Uses: Blood culture, Sterility tests, Continuous culture
methods
Disadvantage – It does not provide a pure culture from
mixed inoculum.
Labelling of inoculated media
Using a grease pencil or marker pen, label inoculated media with the date and the patient’s
number.
Always label the base of a culture plate, not the lid (lids can be accidentally switched). Label a
slope on the underside of the medium so that the wording does not obscure the culture.
A stab culture should be labelled above the level of the agar.
When a plate is to be incubated anaerobically it should be marked ‘An O2’ or when in a carbon
dioxide atmosphere it should be marked ‘CO2’.
Incubation of Inoculated
Media
Incubation of Inoculated Media
A delay in incubation can affect the viability of pathogens especially anaerobes,
pneumococci, meningococci, gonococci, and Haemophilus influenzae.
It can also increase the risk of plates becoming contaminated from small insects and dust.
Uninoculated and inoculated media must be protected from sunlight.
Microorganisms require incubation at the temperature and in the humidity and gaseous
atmosphere most suited to their metabolism.
The length of time of incubation depends on how long an organism takes to develop the
cultural characteristics by which it is recognized.
Incubation of Inoculated Media
Optimum Temperature : at which Bacteria grows best
Temperature Minimum and Maximum temperature: at which growth stops
Range for routine culturing : 35 to 37 oC
Too dry atmosphere can effect the growth and viability of pathogens e-g gonococci is
rapidly killed by dry atmosphere
Humidity
Placing a piece of damp blotting paper in the bottom of a candle jar is recommended while
culturing gonococci
Microorganisms vary in their need for oxygen and use of it as a means of producing energy.
Gaseous
Requirement Strict aerobes, microaerophilic, strict anaerobe, facultative anaerobe, capnophilic /
carboxyphilic
Culturing of Anaerobes
An anaerobic atmosphere is essential for the growth of strict anaerobes such as Clostridium
species, Bacteroides species, and anaerobic streptococci.
Anaerobic incubation also helps to differentiate pathogens and to isolate facultative
anaerobes from specimens containing commensals, e.g. Streptococcus pyogenes from throat
swabs.
The haemolytic reactions of beta-haemolytic streptococci are also more pronounced
following anaerobic incubation.
There are several techniques for obtaining anaerobic conditions. Those
which are more suited for district microbiology laboratories include the
use of:
• Commercially produced sachets containing oxygen removing chemicals. These recently
developed safe technologies do not produce hydrogen and therefore do not require a
catalyst, i.e. they are non-gas generating systems.
• Copper coated steel wool to remove oxygen.
• Reducing agents in culture media
Assignment
• Commercially produced sachets containing oxygen
Read These headings removing chemicals. These recently developed safe
from the Book and
technologies do not produce hydrogen and therefore do not
write in your own
words require a catalyst, i.e. they are non-gas generating systems.
(Page 59-61) • Copper coated steel wool to remove oxygen.
• Reducing agents in culture media
Also write about Recent advances in Anaerobic culture Media
Anaerobic Methods
Production of vacuum
• Incubate the cultures in a vacuum desiccators.
Displacement of oxygen with other gases
• Displacement of oxygen with hydrogen, nitrogen, helium or CO2. Eg: Candle
jar..
Biological method
• Absorption of oxygen by incubation with aerobic bacteria, germinating seeds
or chopped vegetables.
Reduction of medium
• • By using reducing agents – 1% glucose, 0.1% Thioglycolate
Chemical Methods
• Alkaline pyrogallol absorbs oxygen.// Chromium and Sulphuric acid
McIntosh Jar
GasPak
Morphological Identification
Bacteria grown on solid media as colonies
• A colony is defined as a visible mass of microorganisms all
originating from a single mother cell, therefore a colony
constitutes a clone of bacteria all genetically alike.
Different species of bacteria can produce very
different colonies.
WHOLE SHAPE OF COLONY • Varies from round to irregular to filamentous and rhizoid (root-like)
• Can vary from large colonies to tiny colonies less than 1mm = punctiform (pin-
SIZE OF COLONY
point). Measure with a millimeter rule.
EDGE/MARGIN OF COLONY • Magnified edge shape (use a dissecting microscope to see the margin edge well)
• Color of colonies, pigmentation: white, buff, red, purple, etc.
• Some pigments are water-soluble, others are not.
• If you take a large inoculum and place it in a tube of water or saline, do you see
CHROMOGENESIS color?
• Do you see any pigment if the organism is growing in a broth medium?
• Does incubation temperature affect the color?
• Does the entire colony have the color, or is it more like a bull’s eye?
• Is the colony transparent (clear), opaque (not transparent or clear), translucent
OPACITY OF COLONY (almost clear, but distorted vision–like looking through frosted glass), iridescent
(changing colors in reflected light)?
• How much does the colony rise above the agar (turn the plate on end to determine
ELEVATION OF COLONY
height)?
SURFACE OF COLONY • Smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)
• Butyrous (buttery), viscid (sticks to loop, hard to get off), brittle/friable (dry, breaks
CONSISTENCY or TEXTURE
apart), mucoid (sticky, mucus-like)
– Use a plate which has well-isolated colonies. Look at the largest colonies with the naked eye to
determine general shape and chromogenesis.
– Use a dissecting/stereoscopic microscope for more detail. Place the plate RIGHTSIDE UP on the
stage, leaving the petri dish cover ON (Otherwise, your culture will become contaminated.) There
are 2 lenses on scopes—10X and 20X - the black lens knob is on the right side of the head of the
microscope. The magnification is especially helpful for the study of elevation, surface, opacity, size,
and edge. There are 2 lights on these microscopes that you might find helpful, either using one at a
time, or both, or even sometimes without them. Two small black rotating knobs on either side of
the base control the 2 lights, one light from above and one light from below the stage.
– Or you may want to use the Quebec colony counter since it has a magnifying glass, and a light
behind the plate stage. Make sure that the dish is right-side up.
– If you see water condensation on the lid cover, take a Kim Wipe and carefully remove the water
from the cover, then quickly replacing the cover on the dish.
– In order to determine CONSISTENCY, you need to use your inoculating loop or needle to pick up the
colony and determine the consistency of the inoculum material as the loop leaves the agar medium
Identify the colony morphologies
of different species
Staphylococcus aureus: large opaque, round, Streptococcus pyogenes: small translucent Streptococcus pneumoniae: small colonies
creamy, white to yellowish colonies displaying colonies displaying beta-hemolysis on blood with raised edges displaying alpha-hemolysis
beta-hemolysis on blood agar agar on blood agar
Proteus sp.: swarming Serratia marcescens: red
behaviour on blood agar pigmentation: although considered Bacillus cereus: "ground-
characteristic of the species, only glass" colonies displaying
about 10% of specimens produce beta-hemolysis on blood agar
this pigment