MLS 413: BASIC MEDICAL BACTERIOLOGY AND MYCOLOGY
DESIGN AND PREPERATION OF CULTURE
MEDIA
by
Prof. D.E Agbonlahor and Dr. Bankole Henry
Oladeinde
Department of Medical Laboratory Science
School of Basic Medical Sciences
Edo University, Iyamho Edo State
INTENDED LEARNING OUTCOME
• At the end of this class, students should be able to
• Define culture media and discuss its role in diagnostic microbiology
• Describe the evolution of culture media
• List the different types of bacterial culture media and their
application in the medical microbiology laboratory
• List and discuss the constituents of culture media
INTRODUCTION
• Studying the characteristics of microbes requires that they are
grown.
• In the host, microbes have all they need to grow
• One factor that microbes require to grow is nutrient
• Outside the host, they may die if this and other factors are not
met.
• Culture media, tends to provide for the nutritional needs of
microbes outside the host
INTRODUCTION
• Culture media are synthetic or semi-synthetic materials that
holds nutrients necessary for the growth, storage or transport
of micro-organisms or cells.
• The earliest use of culture media dates back to early 19th
century
• The media were essentially beef based broth with varying
composition
• A significant draw back to this method was the inability to
isolate pure culture
INTRODUCTION
• To address this challenge Robert Kock in 1881, tried out a
number of other media such as
• Coagulated egg albumin
• Starch paste
• Aseptically cut slice of potato
• These did not yield good results
• Finally he developed a meat extract complexed with gelatin
• This new techniques was useful in isolating pure culture in that
it was a solid medium
INTRODUCTION
• This discovery led to the birth of Germ Theory of Disease in
1882.
• However gelatin had two major drawbacks
• At temperature above 25oC, it turns into liquid preventing
incubation at higher temperature
• It is easily hydrolyzed by gelatinase, an enzyme secreted by
most micro-organisms.
• Gelatin was later replaced with agar, on the advise of research
assistant to Robert Koch in 1882.
INTRODUCTION
• Derived from seaweed Agarophyte. Agar melts at 85oc and does not
form a gel until it reaches 32-35oc.
• It is clearer than gelatin and resist hydrolysis by bacteria enzymes
• Consist of two polysacharides viz; agarose (70%) and agarpectin
(30%)
• The ability to grow bacteria on solid media gave rise to the word
colony
• Agar provides the opportunity to study the morphology of bacteria,
but does not provide the nutrients required for growth
• coagulated egg is another gelling agents used in Lowenstein Jensen
BASIC COMPONENTS OF BACTERIA CULTURE
MEDIA
• Today there are a number of culture media designed to meet the
nutritional needs of micro-organisms.
• Bacteria/fungi require three main nutrients viz
• A source for carbon (synthesis of cellular constituents)- represents
50% of dry weight of bacteria
• A source for nitrogen (for protein and nucleic acid synthesis)-
accounts for about 14% of dry weight of bacteria
• A source for ATP (cellular activity)
• The others are comprised of electrolytes such as Ca2, Na, K, P etc
BASIC INGREDIENTS OF BACTERIA CULTURE MEDIA
• Carbohydrate
• Peptone
• Mineral Salt
• Buffer
• Agar (sometimes)
• Indicator (sometimes)
CARBOHYDRATE
• Micro-organism require energy to grow.
• Infusion of carbohydrate and their subsequent breakdown
releases this energy
• Common carbohydrates used include
• Glucose
• Lactose
• Fructose
• Mannose
• Sucrose
• Raffinose
CARBOHYDRATE
• Bacteria utilize specific sugar to produce energy
• For instance, bacteria that utilize lactose must express the
enzyme lactase
PEPTONE
• Primitive culture medium was digested with meat proteins for peptones
production.
• Pancreatin, papain, pepsin, ficin, bromelain are often used as digestive
enzymes
• Other sources of protein include fish, casein, soya meal, groundnut meal,
cotton seed etc
• Peptone provide a range of mineral nutrient needed for bacteria growth. E.g
• It is however low on amino-nitrogen concentration and some salt
• These days commercial peptone powder is used as a major constituent of
culture media
MINERAL SALT
• Mineral salts are growth enhancers and are important part of
culture media
• This is provided for by the introduction of ions such as K, Na, Mn,
Mg, Cl. Ca, SO3, SO4 etc in media
• Macro minerals: Na, K, Cl , P, S, Ca, Mg, Fe
• Micro minerals: Zn, Mn, Br, B, Cu, Co, Mo, V, Sr,
• Agar also incorporates some ions in media
BUFFER
• This is necessary to maintain required PH, and prevent
fluctuation due to cell metabolism
• Common substances used as buffer include
• Carboxylic acid compounds
• Phosphate compounds
• Acetates
INDICATORS
• These are substances put in media to detect the presence of
a metabolite/enzyme etc
• Or demonstrate some characteristics of the organism
• Fermentation of specific sugars is detected by indicators in
media
• E.g Phenol, Neutral red, Bromothymol blue etc
• They can also be micro-organisms
• E.g use of sensitive strains of micro-organisms
CULTURE MEDIA
• To effectively treat infectious diseases, it is important that microbial
pathogens are detected in specimen.
• One way of detecting micro-organism is by getting them to grow to
become visible.
• Media that are used to cause such growth in the laboratory are called
culture media
• Seeing micro-organisms will help to:
• Characterize them
• Study their peculiar characteristics
• Aid in developing and determining appropriate therapy for the disease
CULTURE MEDIA
• Taken outside the host bacteria may die
• Culture media must contain
a. All the nutrients that support growth in the host
b. Right proportion of such nutrients
• Several types of culture media support different classes of
micro-organism
CLASSIFICATION OF BACTERIA CULTURE MEDIA
• Based on texture and consistency, bacterial culture meduia are
placed into 3 groups viz:
A. SOLID MEDIA : In addition to nutrients needed for growth contain
agar
• Agar represent about 1.5-2.0% of medium
• Solid culture media are useful in studying bacterial morphology
B. SEMI- SOLID MEDIA : These are sometimes called broths
• They contain agar, but in the concentration of 0.5% or even less
• They are useful in growing microearophilic bacteria and
determination of bacterial motility.
CLASSIFICATION OF BACTERIA CULTURE MEDIA
• LIQUID MEDIA: These have the required nutrient but are void of
gelling agent such as Agar.
• Can be used for demonstration of bacterial motility, fermentation
and biochemical tests.
• Classification based on function
• Based on function or purpose, bacteria culture media can be placed
in the following classes
• BASAL MEDIA: Contains basic nutrients for bacterial growth.
• It is a simple medium that supports non-fastidious micro-
organisms. Example include peptone water, nutrient agar, nutrient
broth
CLASSIFICATION OF BACTERIA CULTURE MEDIA
• ENRICHED MEDIA: In addition to basic nutrients, contains extra
nutritive factors e.g blood, serum. Egg yolk etc. Used to grow
fastidious bacteria.
• They are generally solid media. Examples include blood agar,
chocolate gar, Loefller serum slope, egg yolk medium , yeast
extract etc.
• ENRICHMENT MEDIA: Like enriched media, contain extra
nutritive factors that encourage the growth of bacteria, but are
liquid or semi-solid in nature.
• Small number of bacteria is first grown to large numbers using
this medium. Example is tryptocase soy broth, selenite F broth,
CLASSIFICATION OF BACTERIA CULTURE MEDIA
• SELECTIVE MEDIA: These are media that are used to grow specific
bacteria while inhibiting growth of others.
• They contain inhibitory substances that negate the growth of
unwanted bacteria.
• Inhibitory substances nay be antibiotics, dyes, etc.
• Used when culturing samples from sites with normal flora.
• Examples include Mannitol Salt Agar, Sellenite F agar, Telluriet
Agar , Salmonella Shigella Agar etc
CLASSIFICATION OF BACTERIA CULTURE MEDIA
• DIFFERENTIAL MEDIA: Used to differentiate closely related
bacteria owing to presence of certain dyes or chemicals that cause
bacteria to elicit a particular defining character.
• E.g Eosin Methylene Blue Agar, MacConKey Agar.
• Blood Agar can also be considered differential medium by reason
of its ability to show hemolysis with different bacteria
CLASSIFICATION OF BACTERIA CULTURE MEDIA
• TRANSPORT MEDIA : This is a holding medium when delay in
culture is anticipated.
• It keeps the viability of the bacterium, without allowing their
multiplication
• Stuart media for suspect Neisseria spps positive samples
• Amies transport medium for campylobacter spps
• Cary Blair Transport medium for samples suspected of having
Salmonella, Shigella, Vibrio or Campylobacter
• Sach’s buffered glycerol saline: for samples for bacillary dysentery
• Venkatraman Ramakrishnan medium: for samples for V, cholerea
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