AGAR
Agar is a solidifying agent and a jelly-like substance used in the preparation of the tissue culture
media. Chemically, it’s a gelatinous substance that is extracted from seaweeds. Agar is composed of
two components: agarose, and agaropectin. Agarose is a linear polymer of repeating units of
agarobiose. Agarobiose is a disaccharide that consists of D-galactose and 3, 6-anhydro-L-
galactopyranose.
In 1982, Robert Koch identified it as an important asset to grow bacterias in the lab. Thus, the
manufacturing of bacteriological agar was initiated.
Agar is produced from Gracilaria seaweed. However, it was producing a very poor quality agar which
couldn’t gel properly. This agar was named agroids. Then Yaganwa introduces the solution for this
problem. He transformed the agar obtained from Gracilaria into a strong final product using the
alkaline hydrolysis method.
Now, the two techniques available for agar manufacturing include the freezing-thawing method and
the Syneresis method. In the freezing and thawing method, the extracted agar from algae is frozen in
a freezing tank. Then, they are thawed and strained that makes the agar more concentrated than
before. The syneresis method is an advanced and modern technique of manufacturing agar. In this
technique, the extracted agar is packed in a cloth with a closed mesh. Then, a horizontal hydraulic
pressure is applied to remove water and other impurities from the agar.
The syneresis method is more cost-effective and utilizes lesser energy than the freezing-thawing
method. Agar produced by the syneresis technique has higher purity than the one produced by the
freezing-thawing method. The dry weight extract obtained after pressing is generally 20% in the
syneresis method and 11% in the freezing-thawing method.
Given below is a list of typical gel temperatures extracted from different agarophytes.
S.
Algae Temperature
No.
1 Gelidiella 42-45oC
2 Gracilaria 40-42 oC
3 Gelidium 36-38 oC
4 Pterocladia33-35 oC
TYPES OF AGAR
Different algae produce different kinds of agar. Each agar has distinct characteristics that satisfy
different applications. The agar is solidified because of its agarose content. Agarose has the potential
ability to melt when heated and solidify when cooled. Because of this characteristic, they are termes
“physical gels”. Polyacrylamide polymerization is an irreversible process and they are termed
chemical gels.
Given below is a list of different types of agar that support the different strains of bacterial growth.
S. No. Type of Agar Bacterial studies
1 Blood agar Support growth of most bacteria
2 Luria Bertani (LB) agar Used for routine cultivation of fastidious microorganisms and serve
as a general medium for microbiological studies
3 Chocolate agar Support growth of Haemophilus species and Neisseria
4 MacConkey agar Supports the growth of gram-negative bacteria
5 Nutrient agar To grow different type of bacteria (not all) and some fungi
6 Neomycin agar To culture microorganisms anaerobically
Preparation of media and cultures
Culture media
The method for the preparation of basic microbiology media is given below. In situations where
preparation is uneconomic in time, prepared, sterilized media (liquid and solid) are available from
the major school science equipment suppliers. Sterilization is at 121 °C (15 lb in ˉ²) for 15 minutes.
pH values are 7.0 unless stated otherwise.
Note: Allow 15 cm³ of agar for each Petri dish and 5-10 cm³ of broth for each McCartney bottle. All
cotton wool plugs should be made of non-absorbent cotton wool. Plastic or metal caps may also be
used.
Nutrient agar
Suspend 28 g of nutrient agar powder in 1 litre of distilled water. Bring to the boil to dissolve
completely. Dispense as required and sterilize.
Nutrient broth
Add 13 g of nutrient broth powder to 1 litre of distilled water. Mix well. Dispense as required and
sterilize.
Malt extract agar
Suspend 18g agar powder in 1 litre of distilled water. Bring to the boil to dissolve completely. Add
15g malt extract per litre. Mix well. Dispense as required and sterilize.
Mannitol yeast extract agar
Suspend 10 g agar in 1 litre of distilled water. Heat to dissolve. Add 0.5 g K2HPO4 , 0.2g
MgSO4.7H2O, 0.2 g NaCl, 0.2 g CaCl2.6H2O, 10 g mannitol and 0.4 g yeast extract. Dispense as
required and sterilize.
Mannitol yeast extract broth
As above, without agar.
Glucose nutrient broth
Make up nutrient broth as already directed and add 10 g per litre of glucose.
Sugar peptone water
Add 10 g of peptone, 5 g of NaCl, 5 g of sugar and 20 cm³ of Universal indicator to 1 litre of distilled
water; pH should be 7.4. Dispense as required and sterilize.
Tributyrin agar
Supplied ready for use. Heat to melt and dispense aseptically. May be prepared by adding 1%
tributyrin to nutrient agar.
Glucose yeast extract broth
Add 10 g of peptone, 5 g of NaCl, 3 g of yeast extract to 1 litre of distilled water. Dispense as
required and sterilize.
Glucose yeast extract lemco broth
Add 10 g of Lemco (meat extract) to glucose yeast extract broth.
Milk agar
Make up nutrient agar as above but using only 900 cm³ of distilled water. Dissolve 20 g of dried
skimmed milk in 100 cm³ of distilled water. Sterilize separately. Transfer the milk to the agar
aseptically after cooling to 45-50 °C. Dispense aseptically.
Starch agar
Suspend 15 g of nutrient agar in 100 cm³ distilled water. Bring to the boil to dissolve completely.
Heat 40 g of soluble starch in 100 cm³ of distilled water to form a suspension. Allow to cool and then
mix with the nutrient agar solution. Dispense and sterilize.
Types of culture media used in microbiology Media are of different types on consistency and
chemical composition.
A. On Consistency:
1. Solid Media. Advantages of solid media: (a) Bacteria may be identified by studying the colony
character, (b) Mixed bacteria can be separated. Solid media is used for the isolation of bacteria as
pure culture. 'Agar' is most commonly used to prepare solid media. Agar is polysaccharide extract
obtained from seaweed. Agar is an ideal solidifying agent as it is : (a) Bacteriologically inert, i.e. no
influence on bacterial growth,
2. (b) It remains solid at 37°C, and (c) It is transparent.
3. Liquid Media. It is used for profuse growth, e.g. blood culture in liquid media. Mixed organisms
cannot be separated.
B. On Chemical Composition :
1. Routine Laboratory Media
2. Synthetic Media. These are chemically defined media prepared from pure chemical substances. It
is used in research work. ROUTINE LABORATORY MEDIA These are classified into six types: (1) Basal
media, (2) Enriched media, (3) Selective (4) Indicator media, (5) Transport media, and (6) Storage
media. 1. BASAL MEDIA. Basal media are those that may be used for growth (culture) of bacteria
that do not need enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone
water. Staphylococcus and Enterobacteriaceae grow in these media. 2. ENRICHED MEDIA. The media
are enriched usually by adding blood, serum or egg. Examples: Enriched media are blood agar and
Lowenstein-Jensen media. Streptococci grow in blood agar media.
3. SELECTIVE MEDIA. These media favour the growth of a particular bacterium by inhibiting the
growth of undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey agar,
Lowenstein-Jensen media, tellurite media (Tellurite inhibits the growth of most of the throat
organisms except diphtheria bacilli). Antibiotic may be added to a medium for inhibition.
4. INDICATOR (DIFFERENTIAL) MEDIA. An indicator is included in the medium. A particular organism
causes change in the indicator, e.g. blood, neutral red, tellurite. Examples: Blood agar and
MacConkey agar are indicator media.
5. TRANSPORT MEDIA. These media are used when specie-men cannot be cultured soon after
collection. Examples: Cary-Blair medium, Amies medium, Stuart medium.
6. STORAGE MEDIA. Media used for storing the bacteria for a long period of time. Examples: Egg
saline medium, chalk cooked meat broth
COMMON MEDIA IN ROUTINE USE Nutrient Broth. 500 g meat, e.g. ox heart is minced and mixed
with 1 litre water. 10 g peptone and 5 g sodium chloride are added, pH is adjusted to 7.3. Uses: (1)
As a basal media for the preparation of other media, (2) To study soluble products of bacteria.
Nutrient Agar. It is solid at 37°C. 2.5% agar is added in nutrient broth. It is heated at 100°C to melt
the agar and then cooled. Peptone Water. Peptone 1% and sodium chloride 0.5%. It is used as base
for sugar media and to test indole formation. Blood Agar. Most commonly used medium. 5- 10%
defibrinated sheep or horse blood is added to melted agar at 45-50°C. Blood acts as an enrichment
material and also as an indicator. Certain bacteria when grown in blood agar produce haemolysis
around their colonies. Certain bacteria produce no haemolysis. Types of changes : (a) beta (p)
haemolysis. The colony is surrounded by a clear zone of complete haemolysis, e.g. Streptococcus
pyogenes is a beta haemolytic streptococci, (b) Alpha (a) haemolysis. The colony is surrounded by a
zone of greenish discolouration due to formation of biliverdin, e.g. Viridans streptococci, (c) Gamma
(y) haemolysis, or, No haemolysis. There is no change in the medium surrounding the colony,
Chocolate Agar or Heated Blood agar: Prepared by heating blood agar. It is used for culture of
pneumococcus, gonococcus, meningo- coccus and Haemophilus. Heating the blood inactivates
inhibitor of growths. MacConkey Agar. Most commonly used for enterobac-teriaceae. It contains
agar, peptone, sodium chloride, bile salt, lactose and neutral red. It is a selective and indicator
medium : (1) Selective as bile salt does not inhibit the growth of enterobactericeae but inhibits
growth of many other bacteria. (2) Indicator medium as the colonies of bacteria that ferment lactose
take a pink colour due to production of acid. Acid turns the indicator neutral red to pink. These
bacteria are called 'lactose fermenter', e.g. Escherichia coll. Colourless colony indicates that lactose
is not (3) fermented, i.e. the bacterium is non-lactose fermenter, e.g. Salmonella. Shigella, Vibrio. (4)
(5) Mueller Hinton Agar. Disc diffusion sensitivity tests for antimicrobial drugs should be carried out
on this media as per WHO recommendation to promote reproducibility and comparability of results.
(6) (7) Hiss's Serum Water Medium. This medium is used to study the fermentation reactions of
bacteria which can not grow in peptone water sugar media, e.g. pneumococcus, Neisseria,
Corynebacterium. (8) (9) Lowenstein-Jensen Medium. It is used to culture tubercle bacilli. It contains
egg, malachite green and glycerol. (1) Egg is an enrichment material which stimulates the growth of
tubercle bacilli, (2) Malachite green inhibits growth of organisms other than mycobacteria, (3)
Glycerol promotes the growth of Mycobacterium tuberculosis but not Mycobacterium bovis. (10)
Dubos Medium. This liquid medium is used for tubercle bacilli. In this medium drug sensitivity of
tubercle bacilli can be carried out. It contains 'tween 80', bovine serum albumin, casein hydrolysate,
asparagin and salts. Tween 80 causes dispersed growth and bovine albumin causes rapid growth.
(11) Loeffler Serum. Serum is used for enrichment. Diphtheria bacilli grow in this medium in 6 hours
when the secondary bacteria do not grow. It is used for rapid diagnosis of diphtheria and to
demonstrate volutin granules. It contains sheep, ox or horse serum
(12) Tellurite Blood Agar. It is used as a selective medium for isolation of Cotynebacterium
diphtheriae
