Culture media provides nutrients for bacteria to grow in vitro. Agar is commonly used to solidify liquid media into solid media, allowing for distinct bacterial colonies to form. Specialized media can be selective, differential, or enriched depending on the bacteria being cultured. Proper preparation, storage, and pouring techniques are required to avoid contamination when using culture media in the microbiology laboratory.

1. Media preparation
MBM 350
Mrs Kamanga

2. Introduction
• Bacteria occur as mixed population in nature
• In order to grow bacteria sufficient nutrition must be provided under
appropriate conditions
• It must be grown separately (isolated) on culture media and obtained
as pure culture for study
• Media provides the nutrients required for the bacteria to grow
Liquid media: diffused growth no identification method
Solid media: distinct colonies, easy to identify
• Colony is a macroscopically visible collection of millions of bacterial
cells

3. History of culture media
• Louis Pasteur used simple broths made up of urine or meat extracts
• Robert Koch realized the importance of solid media and used potato
pieces to grow bacteria
• It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse
(who was an assistant to Robert Koch) that agar was used to solidify
culture media
• Before the use of agar, attempts were made to use gelatin as
solidifying agent
• Gelatin had some inherent problems
existed as liquid at normal incubating temperatures (35-37ᴼC)
Digested by certain bacteria

4. Agar
• Solid medium is made by adding Agar
• Agar is obtained from Sea weeds New Zealand
• Contain long chain poly saccharides, inorganic salts and protein like
substance
• Melts at 100ᴼc and sets at 45ᴼc
• No nutritive value
• Not affected by the growth of the bacteria
• 2% agar is employed in solid medium
• Comes as a powder

5. Bacterial Growth Curve
• During typical bacteria growth (growth cycle)bacteria cell divide by
binary fission
• Their mass and number increase in an exponential manner
• Bacterial growth inculture can be separated into at least four distinct
phases;
Lag phase
Log phase
Stationary phase
Death phase

6. Lag phase
• This is period of intense physiologic adjustment involving;
increase in size of cells
increase in metabolic rate
adaptation to new environment
Induction of necessary enzymes
• The length of lag phase depend upon;
The length of lag phase depend upon
Better the medium, shorter the lag phase
The phase of culture from which inoculation was taken
Size or volume of inoculum
Environmental factors like temperature

7. Log/Exponential phase
• The bacterial cell start dividing and their number increase with time
• Bacteria have high rate of metabolism
• Bacteria are more sensitive to antibiotics
Stationary phase
• Rate of multiplication and death becomes almost equal due to:
depletion of nutrient
accumulation of toxic products
sporulation may occur during this stage

8. Decline or death phase
• Population decreases due to death of cells as a result of:
nutritional exhaustion
toxic accumulation
autolytic enzymes

9. Culture media
• Culture media: is a medium (liquid or solid) that contains nutrients to
grow bacteria in vitro
• Nutrient agar: contain 2% agar, can be increased to 6% to prevent
swarming
• Colony: It’s a collection of bacterial cells originating from a single
mother cell. It is the smallest bacterial unit that can be seen with the
naked eye
• Basic requirements of culture media
Nutrients: Energy source - Carbon source - Nitrogen source
Mineral salts: sulphate, phosphates, chlorides & carbonates of K, Mg & Ca
Accessory growth factors: Tryptophan for Salmonella typhi, X & V factors for
H. influenzae
A suitable pH – 7.2 – 7.4

10. Forms of culture media
Solid agar
• Are prepared by adding a solidifying agent (agar 1.5 -2%).
• Prepared mainly in Petri dishes, but also in tubes and slopes.
• After growth the bacterial colonies are visible. e.g. blood agar,
chocolate agar, MacConkey agar
Advantages
Useful in identifying different types of bacteria by seeing shape and color of
colonies
Used to isolate pure cultures
Promote surface growth
Ideal for culture storage
Helpful in the observation of biochemical reactions
Used to make slants, deeps, and plate

11. Cont…
• Semisolid agar (soft agar)
Contains small amounts of agar (0.3-0.7%)
Used to check for motility and also used as a
transport media for fragile organisms
• Liquid (Broth):
Mostly used for biochemical tests (blood culture, Broth culture)
Growth of bacteria is shown by turbidity in medium.
E.g nutrient broth, Selenite F broth, alkaline peptone water

12. Types of culture media
• Based on their consistency;
solid medium
liquid medium
semi solid medium
• Based on the constituents
simple medium- contains basic nutrients for growth
complex medium- They have added ingredients to provide special nutrients
synthetic or defined medium- prepared from pure chemical substances and
its exact composition is known e.g peptone water
Special media
• Based on oxygen requirement
Aerobic
Anaerobic

13. Special media
• Enriched media: Substances like blood, serum, egg are added to the
basal medium used to grow bacteria that are exacting in their
nutritional needs e.g chocolate and blood agar
• Enrichment media: Liquid media used to isolate pathogens from a
mixed culture. Media is incorporated with inhibitory substances to
suppress the unwanted organisms e.g selenite broth for Salmonella,
alkaline peptone water for Vibrio
• Selective media: The inhibitory substance is added to a solid media
e.g MacConkey agar for gram negatives, Lowensten Jensen agar for
Mycobacterium tuberculosis

14. Cont…
• Indicator media: These media contain an indicator which changes its
colour when a bacterium grows in it e.g blood agar, MacConkey agar
• Differential media: Media which has substances incorporated in it
enabling it to distinguish between bacteria e.g MacConkey agar which
distinguishes between lactose fermenters (pink) and non lactose
fermenters (colourless)
• Transport media: Media used for transporting samples. Delicate
organisms may not survive the time taken for transporting the
specimen without a transport media e.g buffered glycerol saline
(entericbacilli). Usually non nutrient soft agar gel

15. Culture methods
• Culture method employed depends on the purpose for which it is
intended;
To isolate bacteria in pure cultures
To demonstrate their properties
To obtain sufficient growth for the preparation of antigens and for other tests
For bacteriophage & bacteriocin susceptibility
To determine sensitivity to antibiotics
To estimate viable counts
Maintain stock cultures

16. Aerobic culture 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.

17. Cont…

18. 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
Antibiotic sensitivity testing
• Lawn cultures are prepared by flooding the surface of the plate with a
liquid suspension of the bacterium.

19. Stroke culture
• Stroke culture is made in tubes containing agar slope / slant
Use;
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

20. 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

21. Liquid cultures
• 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 inocula.

22. Anaerobic culture methods
• Anaerobic bacteria differ in their requirement and sensitivity to
oxygen
• Clostridium tetani is a strict anaerobe (grows at an oxygen tension < 2
mm Hg)
1. Production of vacuum
• Displacement of oxygen with other gases; chemical method or
biological method
• Incubate the cultures in a vacuum desiccators.
• Displacement of oxygen with other gases like hydrogen, nitrogen,
helium or CO2 Eg Candle jar

23. 2. Chemical method
• Alkaline pyrogallol absorbs oxygen
• Chromium and Sulphuric acid McIntosh – Fildes’ anaerobic jar
Consists of a metal jar or glass jar with a metal lid which can be clamped air
tight. The lid has 2 tubes; gas inlet and gas outlet.
Inoculated plates are placed inside the jar and the lid clamped air tight
The outlet tube is connected to a vacuum pump and the air inside is
evacuated
The outlet tap is then closed and the inlet tube is connected to a hydrogen
supply
After the jar is filled with hydrogen, the electric terminals are connected to a
current supply, so that the palladinised asbestos is heated to act as a catalyst
for the combination of hydrogen with residual oxygen.

24. 3. Gaspak
• Commercially available disposable envelope
• Contains chemicals which generate H2 and CO2 on addition of water
• Cold catalyst – permits combination of Hydrogen & Oxygen
• Indicator is used, reduced methylene blue
Colourless - anaerobically
Blue colour- on exposure to oxygen
4. Biological method
• Absorption of oxygen by incubation with aerobic bacteria, germinating
seeds or chopped vegetables
• Reduction of oxygen by using reducing agents; 1% glucose, 0.1%
Thioglycolate

25. Media preparation
• Re-hydrate powder according to manufacturer’s instructions
• Before sterilization, ensure ingredients are completely dissolved,
using heat if necessary
• Avoid wastage by preparing only sufficient for either immediate use
(allowing extra for mistakes) or use in the near future. Normally allow
15-20 cm3 medium/ Petri dish
• Dispense in volumes appropriate for sterilization in the
autoclave/pressure cooker
• Agar slopes are prepared in test tubes by allowing sterile molten
cooled medium to solidify in a sloped position

26. Cont…

27. Pouring a plate
1. Collect one bottle of sterile molten agar from the water bath
2. Hold the bottle in the left hand; remove the lid with the little finger
of the right hand.
3. Flame the neck of the bottle.
4. Lift the lid of the Petri dish slightly with the right hand and pour the
sterile molten agar into the Petri dish and replace the lid.
5. Flame the neck of the bottle and replace the lid.
6. Gently rotate the dish to ensure that the medium covers the plate
evenly.
7. Allow the plate to solidify.
8. Seal and incubate the plate in an inverted position

28. Cont…

29. Storage of media
• Should be stored between 2-8ᴼc immediately upon arrival
• Plates should be stored in the inverted position to prevent moisture
from contacting the surface of the media
• Media should never be exposed to sunlight or UV light, since many
ingredients, especially dyes and indicators, are not stable upon light or
heat exposure
• Media stock or remaining inventory must be rotated to ensure that the
oldest media is used first
• Do not attempt to use media beyond the expiration date stated on
each package

30. Common media used in microbiology lab
• Blood agar
non-selective media contains general nutrients and 5% defibrinated sheep
blood or in some locations, horse blood
This is the most commonly used medium, and supports the growth of the
most common organisms
used to determine various patterns of hemolytic activity
• Chocolate agar
blood agar prepared by heating blood to 85ᴼC until medium becomes brown
or chocolate in color
heating the blood releases both X and V growth factors and also destroys the
inhibitors of V factor
These factors are required for the growth of most species of Haemophilus
and also Neisseria gonorrhoea

31. Cont…
• Mueller Hinton Agar
Rich medium that support the growth of most microorganism
It is commonly used for antibiotic susceptibility testing: disk diffusion
antibiotic susceptibility
• MacConkey agar
MacConkey agar is both selective and differential
It contains bile salts and the dye crystal violet, which inhibit the growth of
gram-positive bacteria and select for gram-negative bacteria.
It also contains the carbohydrate lactose, which allows differentiation of
gram-negative bacteria based on their ability to ferment lactose
Organisms which ferment lactose produce acid end-products which react with
the pH indicator neutral red, and produce a pink color.