Escolar Documentos
Profissional Documentos
Cultura Documentos
Preparation, Dispensing
and Sterilizing of Media
for Cultivation of
Microorganism
GS/M.Sc./FOOD/3608/08
B.K.K.K.Jinadasa
Page 0 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
Introduction
Medium is defined as any substrate or material that will enable microorganisms to grow and
multiply. A common nutrient medium used for culturing microorganisms are exist in three forms
i.e. liquid, semisolid and solid medium. In liquid medium all nutrients are added to water. It is
also called broth e.g. nutrient broth. The liquid medium can be made into solid or semisolid
medium by adding different quantity of solidifying agents like agar-agar, gelatin, silica gel, etc.
Example: nutrient agar, potato dextrose agar, martine rose Bengal agar etc.
The medium should contain all the essential substances required by microorganisms to be
cultured. It should provide carbon, nitrogen, water source in addition to minerals and other
growth factors for the growth of microorganisms. The basal nutrient medium can be
supplemented with different substances such as sugars, proteins, inorganic salts etc. to satisfy the
requirement of a particular organism.
On the basis of chemical components used for preparation of media, it is classified into three
categories.
1) Synthetic media: media in which all the constituents are chemically defined. They are
generally used to study the specific nutritional requirements of different microbes.
2) Complex media: media in which different components and their composition are
incompletely defined. E.g. beef extract used in nutrient medium is chemically complex.
3) Natural medium: substances of natural origin that favours microbial growth are used in
natural media. E.g. milk
1) Simple media: it is a common media used for cultivation of most of the microorganisms.
E.g. nutrient agar
2) Differential media: these types of media are used to distinguish microorganisms that
differ in their specific property. E.g. EMB agar, starch agar, blood agar
3) Selective media: these types of media selectively allow the growth of particular type of
organisms and prevent the growth of the most of other microbes. E.g. martine rose
Bengal agar.
Page 1 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
4) Selective differential media: these type of media allow only a particular type of
organisms to grow that can also be further distinguish based on their property. E.g.
Mc.Conkey agar.
1.1. Preparation, dispensing and sterilization of PDA (Potato dextrose agar) and NA
(Nutrient agar)
Potatoes – 50 g
Agar -5g
Sucrose -5g
Procedure
Potatoes were peeled and cut into small pieces. Out of that 100g of peeled potatoes were taken
Distilled water was added to it and boiled for 20 minutes. Potato extract was filtered through a
muslin cloth to a conical flask.
10g of agar was added to the conical flask heated and was shaken to dissolve.
Then the contents of the conical flask were transferred to a 1litre measuring cylinder and volume
was adjusted to 500ml using distilled water.
8 ml of tartaric acid was added into 500ml of PDA (dissolve 1g of tartaric acid in 10ml of
distilled water and sterilized. 1.6 ml of that solution is added to 100ml of PDA)
PDA media was poured in to Petri plates at 55°C - 60°C, before the media gets solidified.
Page 2 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
Nutrient Agar is a complex medium. It supports the growth of a wide range of microorganisms.
Nutrient media contain all the elements that most bacteria need for growth and are non-selective.
The amino acid source contains a variety of compounds with the exact composition being
unknown.
Beef extract is the commercially prepared dehydrated form of autolysed beef and is supplied in
the form of a paste.
Peptone is casein (milk protein) that has been digested with the enzyme pepsin. Peptone is
dehydrated and supplied as a powder.
Peptone and Beef Extract contain a mixture of amino acids and peptides.
Ingredients
Peptone –0.5 g
Agar- 1.9 g
Procedure
Ingredients were suspended in 100ml of distilled water and boiled to dissolve completely.
Page 3 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
Discussion
When preparing potato dextrose agar, have to add antibacterial agents to inhibit the growth of
bacteria .Chlorumphenicol or tartaric acid can be used as an antibacterial agent.
Containers used for media must have vented tops and should be capable of holding at least 20%
more than the intended volume of medium, to allow for expansion during sterilization. One litre
capped bottles to be very convenient for preparing large quantities, to facilitate cooling,
handling, and pouring. Surface area of the liquid should be large enough to prevent superheating.
Agar does not distribute uniformly when melted. A safe way to ensure a uniform distribution for
pouring plates or tubes is to drop a magnetic stir bar in the flask or bottle, then gently stir the
medium after sterilization, while it cools. Stirring distributes the agar evenly.
1.3.Staining of microorganisms
Introduction
Living Bacteria, like most other cells, are essentially transparent, and must be stained in order to
be easily visualized under the microscope. The specimen must be first spread out on a clean
slide, heated gently to fix it to the slide, and then stained with an appropriate dye. Bacteria tend
to be negatively charged, therefore positively-charged or basic dyes will bind to and stain them.
Staining techniques are widely used to visualise components under the light microscope, for the
differentiation and identification of microorganisms.
Page 4 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
Types of staining
When a single staining-reagent is used and all cells and their structures stain in the same manner,
the procedure is called simple staining procedure. They are also referred as monochrome stains.
This procedure is of two types: positive and negative. In positive staining, the stain (e.g.,
methylene blue) is basic (cationic) having positive charge and attaches to the surface of object
that is negatively charged. In negative staining, the stain (e.g., India ink, nigrosin) is acidic
(anionic) having negative charge and is repelled by the object that is negatively charged, and thus
fills the spaces between the objects resulting in indirect staining of the object. Some of the most
commonly used dyes are methylene blue, carbolfuchsin, and crystal violet. Simple stains allow
one to distinguish the shape (morphology) of the bacteria.
When more than one staining reagents are used and specific objects (e.g., specific
microorganisms and or particular structure of a microorganism) exhibit different staining
reactions readily distinguishable, the procedure is called as differential staining. The most widely
used differential staining in microbiology are Gram-staining and acid-fast staining.
The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 –
1938) who originally devised it in 1882 (but published in 1884), to discriminate between
pneumococci and Klebsiellapneumoniae bacteria in lung tissue. It is differentiating bacterial
species into two large groups (Gram-positive and Gram-negative) based on the chemical and
physical properties of their cell walls. This reaction divides the eubacteria into two fundamental
groups according to their stainability and is one of the basic foundations on which bacterial
identification is built. Gram staining is not used to classify archaea, since these microorganisms
give very variable responses.
These are the dyes which stain only the background, e.g., nigrosin or India ink used either for
observing mucilaginous covering enveloping bacteria (capsules) or certain spores of fungi or
cells of unicellular animals. Negative staining is a technique by which bacterial cells are not
stained, but are made visible against dark background. Acidic dyes like eosin and nigrosin are
employed for this method.
b) Selective Stains.
These stains are used for special purposes, to stain particular parts of the organism such as
spores, metachromatin granules, flagella, nuclei, etc. This type of stain is used to identify
particular cellular structures. For example, a flagellar stain enables us to see the flagellar
filaments which some cells use for movement; a spore stain to detect spores; likewise, a
Page 5 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
capsular stain used to visualise capsule material (generally a polysaccharide coat that some
cells produce).
Cultures of required organisms, Clean glass slide, Inoculating loop and needle, Spirit lamp,
Procedure:
Microscopic slides were washed with detergents and dried. A small clean drop of water was
placed on the slide with the help of an inoculating loop. This water drop was inoculated by a
portion of a colony of the given culture using a sterilized inoculating loop. A thin smear was
prepared by this drop and the needle was flamed. Slide with smear was dried by moving it over
the flame for two or three times. Each fixed smear was covered with approximately five drops
of one of the stains for a designated time as indicated below.
Carbolfuchsin : 15 – 30 sec
After keeping the above designated time, excess dye was removed by washing with a gentle
stream of running tap water and blot-dried using a blotting paper. Stained preparations were
observed under the oil immersion objective of the microscope and significant differences in cell
shape and arrangements were observed.
Bacterial cultures, Inoculating loop, Glass slides, Crystal violet, Gram’s iodine, 95% ethanol
Safranin
Procedure:
Thin heat fixed smears of provided bacterial cultures were prepared. Smears were covered with
Page 6 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
crystal violet (basic dye) for 1 minute and then washed with water for a few seconds and
drained off excess water. Smear was flooded with Gram’s iodine solution (mordant) for 1
minute. Washed with water and drained. Carefully decolorized with 95% alcohol (decolorizing
agent) until no more stain comes away (about 30 seconds). Then the slide was washed with tap
water, drained and covered with counter stain safranin solution for 30 seconds. Slide was washed
gently with water, blot dried and observed under the oil immersion objective.
1.3.4. Results
Simple staining
Page 7 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
Blue colour, coccus shaped cells arranged as chains were observed under the microscope.
Purple colour, coccus shaped cells arranged as chains were observed under the microscope.
Red colour, coccus shaped cells arranged as chains were observed under the microscope.
Gram staining
(a) (b)
Page 8 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
The observed cell appearance was Bluish colouredCoccusand Pinkishcolour Rods (Bacillus).
Generally Streptococcus cells are Gram positive and E. coli are Gram negative found in the
environment.
Most staining procedures are performed on fixed cells. Fixing, or heating the dried cells on the
slide, kills the bacteria and causes them to stick to the slide thereby prevent washing off during
washing steps. This is resulted some artifact on the smear.
The smear preparation should very much concern and too thick layering of staining solution will
not allow lights to pass through the object and dye will crack on drying. Also too thin film will
not give good contrast. Bacteria take up the Gram stain differently because they differ in cell
wall composition. Gram-positive bacteria have a thick cell wall layer. Alcohol does not readily
penetrate to decolorize the cell wall of the previously applied crystal violet stain. Gram-negative
cells have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is
removed from these cell walls that are then stained with the safranin counterstain.
1.4. Isolation of Pure Cultures of Bacteria and Fungi from the Environment.
A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at
each step is constant, resulting in a geometricprogression of the concentration in a logarithmic
fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M... Serial dilutions are
used to accurately create highly diluted solutions as well as solutions for experiments resulting in
concentration curve with a logarithmic scale. As stated earlier, this method is commonly used to
obtain pure cultures of those microorganisms that have not yet been successfully cultivated on
solid media and grow only in liquid media. A microorganism that predominates in a mixed
culture can be isolated in pure form by a series of dilutions.
Page 9 of 12
Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism
1ml from 10 -3 to 10 -5 dilutions were transferred separately into sterile Petri plates. (Three
plates from each dilution) and 15 ml of sterilized nutrient agar medium is poured to each plate.
Loop full of 10 -3 to 10 -5 dilutions were taken and streaked on the solidified nutrient agar and
PDA separately. (Three plates from each dilution).
Page 10 of 12