The document outlines the process of bacterial culturing, detailing various types of culture media, including basic, enriched, selective, and differential media, each serving specific purposes in microbiology. It emphasizes the importance of culture media in isolating and identifying pathogens and the significance of proper techniques for preparing, storing, and incubating these media. Additionally, the document discusses quality control measures and methods for creating anaerobic conditions for cultivating anaerobic bacteria.

1. BACTERIAL CULTURING
DAVID MANYIEL AGOK (DMLT@RHSI)

2. CULTURE
• The term culture refers to the process of growing bacteria
on an artificial medium that supports their multiplication to
form visible colonies. A colony is a group of organisms from
one ancestral unit.
• The purpose of using cultural techniques, is to demonstrate
the presence of organisms which may be causing disease
and when indicated to test for susceptibility of pathogens to
microbial agents.

3. TYPES OF CULTURE MEDIUM
Different categories of culture media used in microbiology can be
categorized into the following classes:
• Basic media
• Enriched media
• Enrichment media
• Selective media
• Indicator media
• Transport media
• Identification media

4. SOLID, SEMI-SOLID AND FLUID CULTURE MEDIUM
• Culture media can also be classified according to their consistence
i.e. Solid, Sem-solid and Fluid
SOLID CULTURE MEDIA
• These are media that are solidified by incorporating a gelling agent
such as agar or gelatin.(Japan/News land type)
• Agar is a polysaccharide extract obtained from seaweed commonly
used to solidify culture media because of its high gelling strength.

5. Cont.................
• Most agars used in bacteriological work produce a firm gel at an agar
concentration of 1.5% w/v.
• The low gelling temperature allows heat sensitive nutrients such as whole
blood to be added safely at 45–50O
C.
• It is added to transport media such as Armies medium at a concentration of
0.4–0.5% w/v to give a semisolid gel.
• Solid media are used mainly in Petri dishes as plate cultures.
• In bottles or tubes they are used as stab (deeps) or slope cultures.
• The purpose of culturing on solid medium is principally to isolate discrete
colonies of each organism present in the specimen.
• This will enable pure cultures to be produced for identification and sensitivity
testing.

6. FLUID CULTURE MEDIA
• These media on the other hand don’t contain a gelling agent.
USES OF COMMON LIQUID MEDIA
• Used for demonstration of carbohydrate fermentation.
• Used for enrichment of few microorganisms e.g. selenite F, selenite U,
alkaline peptone water.
• Used for demonstration of gas production by using Durham’s tube
• Used for demonstration of motility.
• Used for indole production. (peptone water contains tryptophan)
• Used for cultivation of organisms for agglutination test.
• Used for emulsification of organisms for sensitivity testing.
• Some organisms produce a surface growth on the medium in which they
are growing e.g. Vibrio cholerae when growing in alkaline peptone water.

7. BASIC MEDIA / BASAL MEDIA
• These are simple media that support growth of microorganisms
that don’t have special nutritional requirements (Non fastidious)
e.g. Nutrient agar and nutrient broth.
• They are also described as general purpose media
• These media are used to prepare enriched media, maintain stock
cultures of control strains of bacteria and for sub culturing
pathogens from differential or selective media prior to
performing biochemical and serological identification tests.

8. Cont...................
Uses:
• They form a basis for the preparation of enriched media.
• They are used for maintaining stock cultures for certain strength
of bacteria.
• They are used for sub culturing microorganisms from differential
and selective media for other biochemical and serological tests.
EXAMPLES OF COMMON BASIC CULTURE MEDIA
• Basal media include: Peptone water, Nutrient agar and Nutrient
broth

9. 1. Peptone water
• Peptone water is constituted of 1% Peptone and 0.5%.sodium
chloride
• It is mainly used to test for indole formation.
2. NUTRIENT AGAR
• This is a nutrient broth which has been solidified by addition of an
agar (nutrient broth plus gelling agent leads to nutrient agar).
• The commonly used gelling agent is agar. There are 2 types of agar
a. Japanese agar to yield 2% concentration
b. Newzealand agar to yield 1.2% concentration

10. 3. NUTRIENT BROTH
• This is usually prepared from ready to use dehydrated nutrient
broth powder.
• The contents of Nutrient broth are similar to those of Nutrient
agar except that agar is omitted
• It’s preparation and storage is similar to that of nutrient agar
• Used for preparation of other culture media
• It should be of best quality;
There are 3 types of nutrient broth;
• Meat infusion broth
• Meat extract broth
• Digest broth

11. ENRICHED MEDIA
• These are media that have been enriched with ingredients that
are required for growth of fastidious organisms e.g.
Haemophilus influenza, Neisseria species and some
Streptococcus species. Examples of enriched media: Blood
agar, Chocolate agar
• Basic media may be enriched with whole blood or lysed blood,
serum, peptones, yeast extract, vitamins and other growth
factors.

12. • Examples of enriched media
1. BLOOD AGAR
• Most commonly used medium. 5-10% defibrinated sheep or
horse blood is added to melted agar at 45-50°C.
• This media is used for cultivation of organisms that survive on
blood such as Haemophilus influenza which requires factors X
and V for it’s growth that are found in blood
• Blood agar is also used as a differential media as it differentiates
haemolysis exhibited by some haemolytic bacteria.
• Haemolysis is categorized as-β Beta (complete), α-alpha (Partial)
and γ- Gamma (No zones of haemolysis)

13. • Beta haemolysis. The colony is surrounded by a clear zone of
complete haemolysis, e.g. Streptococcus pyogenes is a beta
haemolytic streptococcus.
• Alpha haemolysis (Partial). The colony is surrounded by a zone of
greenish discolouration due to formation of biliverdin, e.g. Viridans
streptococci.
• Gamma (y) haemolysis, or, No haemolysis. There is no change in the
medium surrounding the colony

14. • 2. CHOCOLATE BLOOD AGAR (HEATED BLOOD AGAR)
• This is based on the principle that when blood agar is heated red
cells are lyzed, haemoglobin released and the medium becomes
brown/chocolate in color hence the name chocolate agar.
• It supplies the growth factors required by Haemophilus influenza.
It is also used to cultivate nutritionally demanding pathogens
(delicate) such as N.meningitides and S. pneumonia.
• Heating the blood inactivates inhibitor of growths.
Note: Overheating the media should be avoided as that would
make the media unfit for use.

15. ENRICHMENT MEDIA
• These are fluid selective media which contain substances
that enhance growth of certain bacteria while inhibiting
growth of unwanted ones.
• They are commonly used in the lab to isolate Salmonella
and Shigella species from feacal specimens.
• Examples are:
• Selenite F:
It is an enrichment media used for isolation of salmonella
• Selenite U broth:
It is used for enriching micro organisms in urine.

16. SELECTIVE MEDIA
• These are solid media that contain substances such as bile salts,
antibiotics that inhibit the growth of one organism and encourage
growth of one that is intended to grow.
• A selective medium is usually used when culturing a specimen
from a site that has normal microbial flora to prevent unwanted
contaminants overgrowing the pathogen
• PH of a medium can also make it selective e.g. Vibryo cholera can
be isolated on TCBS agar
• Temperature can also be used to select an organism e.g. Listeria
monocytogens can grow at 4o
c where as other organisms are
inhibited.

17. • Examples of selective media
1. DEOXYCHOLATE CITRATE AGAR (DCA)
• This is a selective solid media. It inhibits growth of most enteric organisms
and enhances the growth of Shigella and salmonella.
2. XLD (Xylose Lysine Deoxycholate).
• It is also selective media used to isolate Salmonella and Shigella species
from stool specimens
3. SS (SALMONELLA-SHIGELLA) AGAR.
• It is a selective medium used to isolate Salmonella and Shigella species.
4. TETRATHIONATE BROTH.
• This medium is used for isolating Salmonella from stool. It inhibits normal
intestinal bacteria and permits multiplication of Salmonella.

18. 5. THIOSULPHATE-CITRATE-BILE-SUCROSE (TCBS) AGAR.
• TCBS agar is a selective medium used to isolate Vibrio cholerae and other
Vibrio species from stool.
• Vibrio cholerae produces yellow colored colonies on a bluish –green
medium after 18hrs of incubation due to fermentation of sucrose in the
medium
6. TELLURITE BLOOD AGAR.
• It is used as a selective medium for isolation of Corynebacterium
diphtheriae.
• Tellurite inhibits the growth of most secondary bacteria without an
inhibitory effect on diphtheria bacilli. It is also an indicator medium as
the diphtheria bacilli produce black colonies.
• Tellurite metabolized to tellurium, which has black colour.

19. INDICATOR (DIFFERENTIAL MEDIA)
• These are media to which dyes or other substances have been added to differentiate micro
organisms.
• Most of the differential media distinguish between bacteria by incorporating an indicator
which changes when color when acid is produced following fermentation of a specific
carbohydrate.
• Examples are:
• 1. M ACCONKEY AGAR
• Most commonly used for enterobac­
teriaceae
• This is a differential media in that it differentiates lactose fermenting organisms from none
lactose fermenting organisms.
• When lactose fermenting organisms grow on MacConkey agar, they ferment lactose leading
to production of lactic acid. Production of lactic acid creates an acidic PH in the medium.
• The acidic PH created in the medium changes the color of indicator (Neutral red) to rose pink
hence lactose fermenting colonies will appear rose pink. Non-lactose fermenting colonies
produce colourless colonies

20. Cont..................
• It is also described as Selective and differential media in that
the bile salt does not inhibit the growth of enterobactericeae
but inhibits growth of many other bacteria.
• Examples of lactose fermenting organisms include: Escherichia
coli, Klebsiella pneumoniae.
• None lactose fermenters include: Salmonella. Shigella, Vibrio
• Organisms which don’t grow on MacConkey include:
Streptococcus species, Pasteurella species, etc

21. • 2. CLED MEDIUM (Cystine Lactose Electrolyte deficient medium)
• This is also a differential medium. It differentiates lactose
fermenting organisms from non lactose fermenters
• Fermentation of lactose leads to the production of lactic acid which
reduces the PH of the medium.
• The acidic PH produced changes the color of indicator
bromothymol blue to yellow hence lactose fermenting colonies
appear yellow
• It is mainly designed for enteric bacteria
• This medium is best prepared from ready to use powder available
most suppliers of culture media.

22. TRANSPORT MEDIA:
• These are mostly semisolid media that contain ingredients that prevent
growth of comensals and ensure survival of aerobic and anaerobic
pathogens when specimens cannot be cultured immediately after
collection.
• Their use is particularly important when transporting microbiological
specimens from health centers to district microbiology laboratory.
• When specimens cannot be cultured soon after collection, may be when
patients from whom the sample has been taken is far from the laboratory
• Examples include Cary- blair for preserving enteric pathogens like Vibrio
and Amies transport media for ensuring viability of gonococci in swab
specimens.
• Alkaline peptone water PH 8.6 for Vibrio cholerae transportation

23. IDENTIFICATION MEDIA
• These are media to which substrates or chemicals are added
to help identify bacteria isolated on primary cultures.
• Examples: Peptone water, urea broth, Kligler iron agar.
• Organisms are mainly identified by change in color of the
medium and, or production of gas.
• Organisms used to inoculate identification media must be first
isolated in pure culture.

24. CHOICE OF CULTURE MEDIA
The choice of culture media to use in microbiology lab depends on:
• The major pathogens to be isolated, their growth requirements and the
features by which they are recognized.
• Whether the specimens being cultured are from sterile sites or from sites
having a normal microbial flora
• Cost, availability and stability of different media
• Training and experience of laboratory staff in preparing, using and quality
control of culture medium
Though a selective medium is more selective than a non selective, it has the
following advantages:
• Often avoids sub culturing,
• Isolates a pathogen more quickly and
• Makes it easier to differentiate and interpret bacterial growth.

25. QUALITY CONTROL OF CULTURE MEDIA
• This involves setting up performance checks with confirmed
standard control organisms and checking for sterility as well.
• Preparing culture media correctly to ensure standardization.
• Labeling of all culture media clearly. If colour cords are used, an
identification chart must be displayed.
• During transportation, ensure that the media are transported under
good conditions. They must be packed well to avoid breakage and
leakages.
• Protect them from moisture, sunlight and excessive heat.

26. Cont..............
• A record should be kept of when the bottle of media is received; its
expiry date should also be noted.
• Test the performance of newly prepared culture media by using
controlled species of bacteria and a record should be kept of
performance test. Inoculate a known organism that is staph aureus on
a prepared media and incubate at 37°C for 18-24 hours. The following
day, study the colonial characteristics of staph aureus. If the colonies
are of staph aureus, pass the media ready for use. If they are not of
staph aureus, prepare a new batch of media.

27. Cont............
• Test for sterility of newly prepared culture media by incubating the
prepared culture media plates at 37°C for overnight then check for
any growth. If growth occurs, it is due to contamination, do not pass
the plates. Instead prepare a new batch.If no growth, store the plates
for use.
• For fluid media, contamination will be shown by turbidity after
overnight incubation of the media.

28. STORAGE OF CULTURE MEDIA
• Store the media in a cool dark place with bottles tightly capped.
• Shelf life can go up to years provided if there is no change in the volume
or appearance of the media to suggest contamination.
• Plate media should be stored at 2-8°C in a sealed plastic bag to prevent
drying.
• Plates must not be exposed to sunlight.
• Unused plates should not be left on the bench for overnight to prevent
contamination and deterioration.
• Most slopes and fluid media can be stored at room temperature between
20-28°C.
• New batches of media should be dated and stored separately from the
earlier batches.

29. INCUBATION METHODS
• Inoculated media should be incubated at right atmosphere in
terms of temperature, humidity and gaseous atmosphere that
is most suitable for their metabolism.
• It is important to incubate the inoculated plates as soon as
possible as delay would affect viability of pathogens especially
anaerobes, pneumococci, Meningococci, Gonococci and
Haemophilus influenza. Delay would also expose the plates to
the risk of contamination by small insects and dust.

30. Cont.........................
• Both inoculated and un inoculated plates must be protected from
sun light.
• The length of time of incubation depends on how long an
organism takes to develop the cultural characteristics by which it
is recognized.
• Incubation methods are either aerobic or anaeric.

31. AEROBIC/CULTIVATION INCUBATION:
• This involves processes of growing bacteria in an oxygen enriched
atmosphere.
• The organisms vary in their requirement for oxygen. Some are
strict aerobes, some are facultative, while some require only
trace amounts between 3-10% ( micro aerophilic)
• The inoculated media is incubated at 37O
C in anaerobic incubator
• The period of incubation varies though most will give good
growth in 24 hrs.

32. ANAEROBIC CULTIVATION/INCUBATION
• This involves the process of cultivating bacteria in an oxygen free
atmosphere
• Anaerobes vary in their degree of survival in anaerobic atmosphere;
the concentration of oxygen will depend on the targeted bacteria
which may vary from one organism to the other.
• Strict anaerobes such as Clostridium species, Bacteriodes species
and anaerobic Streptococci survive under such an environment
• The haemolytic reactions of beta-haemolytic Streptococci are also
more pronounced following an aerobic incubation.

33. METHODS OF OBTAINING ANAEROBIC CONDITIONS
1. Use of anaerobic jars with gas generating kit.
• There are several forms of anaerobic jars but all based on the same principle that
oxygen from the chamber reacts with hydrogen from a cylinder to form water.
• Some jars contain chemicals whose reaction results into formation of carbon
dioxide and hydrogen that will replace the oxygen volume.
2. Use of twin plates
• In this method, two organisms are inoculated on different culture plates. One
being aerobe and the other anaerobe.
• The two plates are then sealed together with a tape without their lids and
incubated at 37O
C
• The aerobe will the utilize all the oxygen in the environment hence creating
anaerobic environment.

34. Cont...............
3. Use of a media with reducing agents. Such media include:
• Thioglycollate broth: This medium contains a reducing agent-
sodium thioglycollate and the indicator methylene blue which
indicate that the medium has been reduced.
4. Use of iron nails:
• This is a simple way of creating anaerobiasis in litmus milk
medium, peptone water media by using an iron strip or iron nail
to remove the oxygen.
• The strip or nail is sterilized by a flame and is dropped into the
medium while still hot.The medium is then left to cool before use.

35. Cont..............
• Use of anaerobic glove box: (Closed chamber with sealed –in gloves)
• This is a self contained anaerobic system that allows a microbiologist to process
specimens and carry out other microbiological techniques to isolate anaerobic
organisms without exposure to air.
• The Nitrogen, hydrogen and carbon dioxide replaces the air in the box chamber
• 6. Use of copper coated steel:
• This system uses steel wool which is activated immediately before use by dipping it
in acidified copper sulphate solution.
• The metallic copper on the surface of the iron rapidly absorbs oxygen
• The plates can be incubated in a plastic bag.
• An aerobic conditions are obtained more rapidly by removing some air before
sealing the bag.
• A source of carbon dioxide and the indicator to check for anaerobiasis are added to
the bag.

36. CULTURING IN CARBON DIOXIDE
• A carbon dioxide enriched atmosphere is required for the growth of
Neisseria gonorrhoeae, Neisseria meningitidis, Brucella species, and
Streptococcus pneumoniae.
• Carbon dioxide enriched environment can be obtained by:
1. use of commercially prepared carbon dioxide gas-generating systems.
2. Use of airtight jar or tin with a lighted candle.
As the candle burns, the oxygen content is reduced leaving a carbon
dioxide content of 3–5% by the time the candle is extinguished
• 3. Use of chemicals to generate carbon dioxide
• A mixture of sodium bicarbonate and tartaric acid can create
acarbondioxide enriched atmosphere.

37. • Assignment;
• Mention ways how you can ensure aseptic techniques in microbiology
laboratory
• Explain how you can ensure discrete colonies during bacteriological
culturing

38. The end.........