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Last Update: 4 November 2017 Part - II

M - 64
Bacterial Culture
Culture or cultivation of bacteria under artificial conditions viz. optimum temperature or under optimum gaseous
atmosphere & by providing appropriate metabolic substrates in the form of media.

Purpose of Bacterial Culture


i) Diagnosis of infectious disease.
ii) Isolation of bacteria.
iii) Identification of bacteria (species + strains) in conjunction with biochemical reactions & serology.
iv) Antibacterial drug susceptibility tests.
v) Preparation of antigens / type specific antisera in serology.
vi) Follow-up cultures after a course of antibiotics.
vii) Viable count of bacteria.
viii) Preparation of stock-cultures for experimental bacteriology.
Requirements Types of organisms Definition
Energy Chemotrophs Rely on chemical compounds for energy
Phototrophs Utilize radiant energy (light)
Electron Lithotrophs Can use reduced inorganic compounds as electron donor.
Organotrophs Can use organic compound as electron donor.
Carbon Autotrophs CO2 is used as major, or even sole source of carbon
Heterotrophs Require organic compounds as carbon source

All living organisms require vitamins and vitamin like compounds. These function as coenzymes. Some
bacteria are capable of synthesizing their entire requirement of vitamin from other compounds, but others cannot do so
will not grow unless the required vitamins are supplied.
Except for certain ecological studies where bacterial population are examined in there natural habitats,
bacteria are usually cultivated and studied under laboratory conditions. Numerous media (singular medium) have been
developed for bacterial cultivation. Because the nutritional requirements of bacteria vary widely, there are great
differences in the chemical compositions of the media used in the laboratory.
Chemically defined media are needed for cultivation of autotrophs, certain complex raw materials such as
peptones, meat extracts and yeast extracts are used. Agar is used as a solidifying agent when a solid medium is
desired. These relatively simple liquid and solid media that support the growth of many common heterotrophs are
nutrient broth and nutrient agar.

Nutrient broth Nutrient agar


Beef extract 3 gm. Beef extract 3 gm.
Peptone 5 gm Peptone 5 gm
Water 1000 ml Agar 15 gm
Water 1000 ml

The addition of yeast extract to each of the formulas improves the nutrient quality, since yeast extract contains
several of vitamins and other growth prompting substances. Other complex supplements such as bovine rumen fluid,
animal blood, blood serum or extract of plant and animal tissues may be required for the cultivation of some bacteria
and they are called fastidious heterotrophs.

Culture media An artificial environment with essential nutrients at optimum quantity & at optimum pH which
helps bacterial growth in vitro. Most bacterial are heterotrophic in nature. They need organic materials & special
growth factors in media for their multiplication.

Constituents of Basal culture media


General laboratory media
1. Water source of H2 & O2.
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2. Electrolyte NaCl other electrolytes.
3. Peptone.
Peptone Complex mixture of partially digested proteins prepared from animal or vegetable proteins by enzymatic
action.
Commercial peptone = Bactopeptone = Peptones + proteases + polypeptides + amino as + inorganic Salts (phosphates,
minerals (K, Mg) + Accessory growth factors (Riboflavin)
4. Meat extract, yeast extract : Contain protein degradation products + carbon + inorganic Salts + certain
growth factors. Commercially available as lab-lemco.
5. Blood (Enriches media) Use 5-10% defibrinated horse / sheep blood; sometimes serum also required.
6. Agar derived from sea weed (Algae Geladium sp.)
Contains mainly carbohydrate (complex polysaccharide) + small amt. of protein like material +
sometimes traces of long chain fatty as + variety of impurities (inorganic salts)
Usage 2 3% concentrated as dried fibres as well as powder.
Melts at 95C; usually solidifies at 42C.
Acts merely as a solidifying agent of culture medium doesnt provide any nutrition to bacteria.
Composition varies on its source (New Zealand agar = 2 x jellifying property of Japanese agar)
Pathogenic bacteria cant metabolise agar.

Types of Media Based on nutritional factors / Composition + purpose Simple (basal) media simplest medium
routinely used in laboratory diagnostic purposes.
1% peptone + 0.5% NaCl + distilled H2O + pH 7.4-7.5 = Peptone water +1% meat extract nutrient Broth +
2-3% agar nutrient agar
Nutrient broth reduced concentration upto 0.2 0.4% agar enable motile bacteria to form spreading or
swarming growth

Nutrition Characterization of Bacteria


Bacteria Energy Electron donor Carbon for assimilation
Phototrophic Chemotrophic Lithotrophic Organotrophic Autotrophic Heterotrophic
Chroamatium okenii + + +

(anaerobic
+ + +
Rhodospirillum condition)
rubrum
(aerobic
+ + +
condition)
Nitrosomonas europaea + + +
Desulfovibrio desulfuricans + + +

Pseudomonas (H2 supplied) + + +


Pseudoflova
(no H2
+ + +
supplied)
Escherichia coli + + +

Essential elements required for organisms are as follows


Elements Source Functions Remark
H2 & O 2 H 2O Synthesis of bacterial structural components like O2 serves as terminal
carbohydrates, lipids, proteins. electron acceptor.
C CO2 & different organic Provides energy by the formation of ATP.
substances
N2 Mainly NH3-Salts, NO3, Components of proteins and nucleic acids. NH3, mitrates etc, H2S
NO2 salts serve as electron
donor
P Always assimilated as free Components of nucleic acids, ATP, coenzyme,
inorganic phosphate. NAD, flavins
S Sulphate (SO4) reduced to Forms part of the structures of several coenzymes,
H 2S amino acids (cysteine, methionin)
K Major cataion, activates various enzymes.
Ca
Mg
Fe Electron carrier in electron transport chain Halophilic bacteria
require high
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concentration of Na.
Trace elements Activate and stabilize variety of enzymes.
e.g. Cu, Mn,
Co, Mo, Zn

Media for Characterization of Bacteria


Specific kinds of media are used for determining the type of growth produced by bacteria, as well as to
determine their ability to produce certain chemical changes.
Semisolid media, prepared with agar at concentration 0.5% or less, have a soft custard like consistency and
are useful for the cultivation of micro aerophilic bacteria.

Preparation of Media
Some naturally occurring substances are used for the cultivation of bacteria. Notable among these is milk,
usually skimmed rather than whole. Such natural materials are merely dispensed into tubes or flasks and sterilized
before use. Media of the nutrient broth or nutrient agar type are prepared by compounding the required individual
ingredients or, more conveniently, by adding water to a dehydrated product which contains all the ingredients.
Practically all media are available commercially in powdered form. The preparation of bacteriological media usually
involves the following steps;
1. Each ingredient, or the complete dehydrated medium, is dissolved in the appropriate volume of distilled water.
2. The pH of the fluid medium is determined with a pH meter and adjusted if necessary.
3. If a solid medium is desired, agar is added and the medium is boiled to dissolve the agar.
4. The medium is dispensed into tubes or flasks.
5. The medium is sterilized, generally by autoclaving. Some media (or specific ingredients) that are heat-labile
are sterilized by filtration.

I. On the basis of the application of function, media may be classified as follows:

1. Selective media
These media provide nutrients that enhance the growth and predominance of a particular type of bacterium
and do not enhance (and may even inhibit) other types of organisms. Selectivity is accomplished by (i) high salt
concentration, (ii) absence of growth factors, (iii) extremes of pH, (iv) presence of special inhibitory substances. For
instance, a medium in which cellulose is the only carbon source will specifically select for or enrich the growth of
cellulose-utilizing organisms
Characteristic features
Solid always, Contain additives which enhance growth of desired organisms or strain by inhibiting growth of
all other organisms.
Facilitates isolation of a partially bacteria species from a mixed inoculums, e.g., Bile Salt Agar favours growth
of only Cholera vibrios as inhibits growth of normal microbial flora of intestine.

Selectivity accomplished by :-
i) High salt concentration
ii) Absent or presence of essential growth factor
iii) Extremes of pH or Redox potential
iv) Proliferation of species inhibitory substances.
Eg, DCA Deoxycholate Citrate Agar, Macconkeys agar, Blood tellurite agar, Lowenstein Jensen medium,
TCBS agar, Bile salt agar etc.

2. Differential Media
Certain reagents or supplements, when incorporated into culture media, may allow differentiation of various
kinds of bacteria. For example, if a mixture of bacteria is inoculated onto a blood-containing agar medium, some of
the bacteria may haemolyze the red blood cells, others do not. Thus one can distinguish between haemolytic and non
haemolytic bacteria on the same medium. Some indicator substances incorporated in (e.g. neutral red or bromothymol
blue) or any other reducing substances mixed with media may indicate differentially the presence of differentially
coloured colonies of the organisms.

Assay media
Media of proscribed compositions are used for the assay of vitamins, amino acids and antibiotics.

3. Synthetic media
Synthetic media prepared from pure chemicals. Specific nutrients as well as substrates as and when required
added separately into this medium. This medium thus prepared from the product of known composition. Such
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types of media employed generally in experimental & research work as for example specialized
bacteriological techniques like microbiological assays.
Synthetic media are of two types
a Simple synthetic media: it support growth to non-parasitic heterotrophy (C source as Glucose,
lactate, N source as inorganic : NH4Cl, phosphate or SO4)
b) Complex synthetic media: it favour growth of more exacting bacteria. Such types media contains
extra amino acids, pyruvic acid, growth factor, eg. Dubos medium (= twenty eight amino acids +
casein + hydrolysate + Bovine serum albumin (BSA) + Mineral salts)

Complex Media
Enriched media
Solid Media Blood agar, Serum agar, Chocolate agar, Loefflers serum media, Hydricols fluid agar .
Allows growth of fastidious orgs pr. of specific nutritive additives (growth factors) such as haemin, cysteine, blood,
serum, etc. eg. i) Ascitic fluid or hydrocoele fluid to basal media to meet nutritional demands of more exacting
bacteria. ii) Growth of Francisella in chocolate agar.

Enrichment media Always liquid, favours multiplication of a partial species of bacteria either by
i) incorporating special substances which are selectively favours its growth (through enriching the medium)
ii) or incorporating special substances which are inhibits its competitors
Eg, Selenite broth, tetrathionate broth, alkaline peptone water, Robertsons cooked meat medium.

General Purpose medium


Capable to detect most aerobic & facultatively anaerobic organisms.
e.g. sheep blood agar
Composition Nutrient agar + 5-10% sheep blood
Most widely used in Laboratory General culture, streptococcus
Commonly for general isolation of micro organisms directly from initial specimens inoculated into
agar.

Indicator medium :
Some indicator substances incorporated (eg. neutral red or bromothymal blue indicates visible changes
in media) or any reducing subs may be incorporated too (eg. potassium tellurite)
Colour of indicator in the medium changes with bacterial growth as a result of particular and distinctive
metabolic activities of a particular organisms.
Used to detect colonies of particular bacteria sp. or strain by virtue changes in the media.
Eg. sugar fermentation fubes, Mac Conkeys media, DCA media.
When Salmonella typhi grown in Wilson & Blair medium containing sulphite bacteria colonies show block
metallic sheen due to reduction of sulphite to sulphide.

Differential medium
Apart from helping growth of a particular genus of bacteria, helps differentiation into various sp / strains.
Certain substances help to distinguish differing properties of different bacteria.
Eg, MacConkays agar medium also an indicator medium.
Eg, Tellurite media differentiates C. gravis, C. mitis, C. intermedias
Blood agar differentiates types of streptococci on basis of haemolysis.
Contains peptone, agar, lactose, sodium taurocholate & natural red.
Lactose fermenters form pink colonies
Non-lactose fermenters produce colourless or pale colonies.
Transport medium / carrying media
To protect & preserve delicate pathogens (protect exclusively in specimens faeces, throat swab) during
delay in transport, special media devised :
a) (Stuarts transport medium for urethral discharge (Gonococci & non-clostridial aerobes).
Semi-solid non-nutrient agar thioglycollic agar (red agent) + electrolytes.
b) Glyceralsaline transport medium Glycerol + NoCl + Di-sodium hydrogen phosphate + phenol red
For stool dysentery bacilli.
c) Bile peptone transport Media Trypticase / good peptone + NaCl + Na taurocholote, H2O (pH 8.5)
For stool / faeces / rectal swab Cholera vibrios.

Sugar medium
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Standard sugar media used for biochem. Tests
Contain 1% sugar concerned in peptone H2O + endicator (Andrades indicator, 0.05% a fuchsin in N NaOH)
Glucose, sucrose, lactose & mannitol routinely employed for sugar fermentation tests.
Certain exacting orgs. req. serum for growth, eg. Hisss serum sugars for pneumococcus.

Anaerobic culture media
To cultivate anaerobic organisms.
These are nutritionally enriched + reproduced of molecular O2 for better growth of anaerobes from patient
specimens.
Eg, Robertsons cooked meat media, Thioglycollate media

Mycobacterial culture media Special media only mycobacterium grow. Eg. egg based Lowenstein Jensen
made., Middlbrook broth
Dehydrated media
Prepared from dehydrated ingredients manufactured by commercial farm
Composition with different ingredients mentioned on label of container.
Simply reconstituted in dist H2O + sterilized in lab before use.
Nowadays much used.
Useful for small lab + time-saving.

II. Types of Media based on state


A. Liquid Media
Dispensed in tubes with cotton-wool stoppers or screw-capped bottles
Growth recognized by uniform turbidity of liq-media.
+ flocculent / flaky growth throughout.
+ pellicle on surface or sediment at bottles.
Uses
i) Specimen inoculation eg blood culture bottles.
ii) Recovery of fastidious orgs eg Robertsons cooked meat broth (anaerobes) Kirschaers broth
(Mycobacteria)
iii) Metabolic reactions Urease broth, peptone water.
iv) Antibiotic susceptibility testing Min. inhibitory concentration performed in serially diluted broth
suspensions of antibacterial drugs.
v) Presumptive bacterial count in H2O sample.
Names i) Peptone water ii) Nutrient broth iii) Glucose broth iv) Sugar tubes
v) Robertsons cooked meat media

Advantages of Liquid Media Disadvantages


i) Promote growth of orgs when scanty in i) Not possible to estimate relative nos. of different sp
inoculation / clinical material. Eg blood culture. originally practiced is the clinical material.
ii) Exotoxin can be extracted. ii) Isolation in pure forms not possible from a mixture
of organisms.
iii) Motility study possible. iii) Colony pattern not well marked
iv) Dilution effect : if clinical specimen contains iv) Gram stain appearance of Bacteria grown on liquid
antibiotics or other inhibitory subs these are media may differ from identical orgaisms grown
diluted on inoculation into larger volume of liquid on solid media.
media thus allowing bacteria growth. Sedimentation of bacteria not possible.
v) Special components (eg.- sugars) used in
biochem reactions to identify bacteria.
vi) Enrichment media are liquid Media with inhibitors
to suppress growth of contaminants & encourage
growth of a specific bacteria.
vii) Large inoculation can be tested eg. gauge for
sterility test.
viii) Presumptive bacteria count in H2O sample made in
liquid medium.

B. Solid Media Mainly for first degree or initial isolation of organisms, these are dispensed in tubes or slobes,
screw capped bottles or in plates, or in Petri dishes.
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Advantages of Solid Media Disadvantage
i) Colony pattern well marked separate colony form Doesnt promote growth of scanty orgs.
ii) Identify of orgs easy from study of colonial morphology
iii) Isolation of pure culture possible by picking isolated
bacteria. Colonies + sub culturing into fresh med regd for
full identify of bacteria.
iv) Quantitative bacteria Count relative proportion of different
bacteria species possible,
Names
i) Nutrient agar ii) Agar-agar
iii) Blood-agar iv) Chocolate agar
v) Chocolate agar vi) Loefflers serum media.
vii) Dorsets egg. Media viii) Macconkeys media
ix) Mac Conkeys media x) Deoxycholate citrate agar
xi) Tetrathionate broth & selenite Fewichment Media

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