In actual practice, the selective media specifically favour the growth of particular microorgan-isms. MacConkey’s agar medium was introduced first and foremost in the year 1905, so as to isolate Enterobacteriaceae from such sources as : urine, faeces, foods, and water.
SELECTIVE
AND DIAGNOSTIC MEDIA
A survey
of literature has adequately established that there exists varying degree of
abilities to carry out the proper fermentation of ‘carbohydrates’, glaring differences in the ‘pyruvic acid metabo-lism’ utilized to have a clear-cut
distinguished features of A. aerogenes and E. coli, varying responses of bacteria to different inhibitors etc.
; and the ensuing exploitation of these critical differences may be expatiated
by the judicious usage of selective and
diagnostic media.
In actual
practice, the selective media
specifically favour the growth of particular
microorgan-isms. MacConkey’s agar medium was introduced first and foremost in
the year 1905, so as to isolate
Enterobacteriaceae from such sources as : urine, faeces, foods, and
water.
Importantly,
the various ingredients incorporated in the above MacConkey’s medium play a definitive role as stated below :
Bile Salts : invariably function as a ‘natural
surface-active agent which does not inhibit the growth of the Enterobacteriaceae,
but certainly prevents the growth of Gram-positive bacteria that are generally
available in the material under investigation.
Lactose : Production of acid from lactose by the help of two organisms, namely : A.
aerogenes and E. coli exert their action on this
medium thereby changing the original colouration of the indicator, besides adsorbing the said indicator to a certain extent
around the growing bacterial cells.
Microorganisms : These may also be selected by incubation in the presence of nutrients which they may consume specifically.
Example : Isolation of cellulose-digesting microorganisms may be accomplished by using a medium containing only ‘cellulose’ as a particular source of carbon and energy.
Salient Features : The
salient features of selective media are
as stated under :
(1) The
microorganisms that specifically cause typhoid
and paratyphoid fever*, and bacil-lary
dysentry** fail to ferment ‘lactose’
; and, therefore, the resulting colonies of these microbes distinctly appear to be transparent absolutely.
(2) Besides,
MacConkey agar there are also two
other highly selective media viz., eosin-meth-ylene blue agar, and
endo agar that are employed widely and exclusively for the detection of E.
coli (most dreadful faecal organism) and allied bacteria present in water
supplies, food products etc., which essentially contains dyes that would
critically suppress the growth of Gram-positive
organism e.g., staphylococci.
(3) Several
accepted modified variants of MacConkey’s medium do exist viz., bile salts are
duly replaced with pure synthetic
surface-active agent(s).
(4) Selectivity
of MacConkey’s medium could be further enhanced by the addition of certain specific inhibitory dyes e.g.,
neutral red and crystal violet.
(5) Importantly,
the MacConkey agar medium serves both differential*
and selective, because it
predominently contains lactose and neutral red dye whereby the particular
lactose-fer-menting colonies distinctly appear pink to red in colour, and are distinguished from the ‘colonies of nonfermentors’ quite
easily.
There are
certain other selective media that
are invariably prepared by the addition of quite a few highly specific
components to the corresponding culture medium which may allow the growth of
one ‘group of microbes’ while
suppressing growth of some other groups. A few typical examples of such ‘selective media’ are as given under :
(a) Salmonella-Shigella Agar [SS-Agar]. It is
exclusively used to isolate both Salmonella
and Shigella species. In fact, its ‘bile
salt mixture’ inhibits the growth
of several cardinal groups of coliforms in particular. Importantly,
the Salmonella and Shigella species give rise to al-most colourless colonies by virtue of the
fact that they are not capable of fermenting lactose. In fact, lactose-fermenting
microorganisms shall produce pink-colonies mostly.
(b) Mannitol-Salt Agar [MS-Agar]. It is
solely employed in the isolation of staphylococci.
The relatively high-level of selectivity is usually accomplised by the high
salt concentration (~ 7.5%) which specifically retards and checks the growth of
several groups of microbes. It is, however, pertinent to state here that the
presence of mannitol in the MS-Agar
medium distinctly aids in the clear-cut differentiation of the ‘pathogenic staphylococci’ from the ‘nonpathogenic staphylococci’ due to
the fact that the former augments fermentation of mannitol to yield ‘acid’
whereas the latter fails to do so.
(c) Bismuth-Sulphite Agar [BS-Agar]. BS-Agar medium was duly
developed in the 1920s solely for the identification of Salmonella typhi, especially from the stool and food speci-mens. It
has been duly proved that S. typhi
reduces the ‘sulphite’ anion to the
corresponding ‘sulphide’ anion,
thereby giving rise to distinct apparently visible black colonies** having a
specific metallic sheen (lustre). On a rather broader perspective BS-Agar medium may also be extended to
identify the presence of S.typhi in
urine, foods, faeces, water, and phar-maceutical products. Generally, the BS-Agar comprises of a buffered
nutrient agar contain-ing bismuth
sulphite, ferrous sulphate, and brilliant green.
Observations. Following are some of the
cardinal observations, such as :
(1) E. coli*** gets usually inhibited by the
presence of brilliant green at a
concentration of 0.0025% (w/v) ;
whereas, S. typhi shall attain growth
luxuriantly.
(2) Bismuth sulphite may also
exert an inhibitory effect to a certain extent upon the E. coli.
In
addition to the ‘selective and
diagnostic media’ one may also come across such types of media as :
(i) Differential
media,
(ii) Enrichment
media, and
(iii) Characteristic
media.
The above
mentioned three types of media shall
now be discussed individually in the sections that follows :
The differential media usually refers to
the incorporation of certain specifc
chemicals into a medium that may eventually give rise to diagnostically
useful growth or apparent change in the medium after the proper incubation.
A few
typical examples are as discussed under :
The EMB-Agar media is employed exclusively
to differentiate between the ‘lactose
fermenters’ and the ‘non-lactose-fermenters’.
In-fact, the EMB-Agar media
essentially comprises of : lactose, salts, and two dyes viz. eosin and methylene blue. From the observations the following inferences may
be drawn :
(a) E.
coli (a ‘lactose fermenter’) :
will produce either a dark colony or one that has a metallic sheen, and
(b) S.
typhi (a ‘nonlactose fermenter’) :
shall appear as an absolute colourless colony.
It has
already been discussed under Section 3.
It is
invariably used to enhance the overall yield of Salmonella and Shigella
species in comparison to other microbiota.
It has been observed that the presence of relatively
high bile salt concentration inhibits the general growth of Gram-positive
microorganisms specifically. Besides, HE-Agar also retards (or slows down) the
growth of several coliform strains.
It has
been amply demonstrated and established the critical and judicious
incorporation of serum, blood, or extracts to the particular ‘tryptic soy agar’ or broth shall enormously augment the much
desired growth of a large number of most ‘fastidious
microbes’. In actual practice, however, these media are largely employed to
isolate primarily the microorganisms from a host of ‘biological fluids’ such as : cerebrospinal
fluid, pleural fluid, wound abscesses, and sputum. A few typical
examples are as stated under :
The
critical addition of ‘citrated blood’
to the prevailing ‘tryptic soy agar’
renders it to afford variable haemolysis,
that in turn allows the precise differentiation of certain species of microorganisms. It is, however, pertinent to state
here that one may observe these distinct haemolytic
patterns on blood agar. A few such typical variations are as stated under :
(a) α-Haemolysis. It may be observed due to the
formation of greenish to brownish halo** around the colony e.g., streptococcus gardonii
and streptococcus pneumoniae.
(b) β-Haemolysis. It represents the virtual complete haemolysis of blood cells
thereby giving rise to a distinct clearing
effect around growth in the colony e.g.,
Staphylococcus aureus and Streptococcus pyogenes.
(c) Nonhaemolytic Pattern. In this
particular instance practically no change occurs in the me-dium e.g., Staphylococcus epidermidis and Staphylococcus
saprophyticus.
Interestingly,
the ‘chocolate agar’ is specifically
made from ‘pre-heated blood’ that
essentially caters for the requisite and necessary growth factors desired urgently to support the bacterial growth e.g.,
Haemophilus influenzae and Neisseria
gonorrhoeae.
The very
purpose and extensive utility of the so-called ‘characteristic media’ are to test mi-crobes for ascertaining a few
highly specific metabolic activities,
products, or their ensuing require-ments.
Following
are some of the typical examples,
namely :
The TSI-Agar usually comprise of : lactose,
sucrose, glucose, ferrous ammonium sulphate [(NH4)2Fe(SO4)2],
and sodium thiosulphate [Na2S2O3]. In actual
practice TSI-Agar is solely used for
the critical identification of enteric
organisms* ; and are broadly based upon their inherent ability to attack
the chemical entities viz., glucose,
lactose, or sucrose and thus are responsible for liberating ‘sulphides’ from ferrous ammonium
sulphate or sodium thiosulphate.
The
various typical examples of TSI-Agar are as stated under :
(a) Citrate Agar. It contains sodium citrate [C6H5Na3O7],
which serves as the exclusive source
of carbon ; whereas, the ammonium
phosphate [(NH4)3PO4] as the sole source of nitrogen. The citrate agar finds its usage to
differentate the ‘enteric bacteria’ on
the basis of ‘citrate utilization’.
(b) Lysine Iron Agar [LIA].
Importantly, LIA is solely employed to differentate microorgan-isms which may
either cause deamination or decarboxylation the amino acid lysine. Because, LIA comprises of lysine that predominantly and
exclusively allows enzyme detection
; whereas the pres-ence of ferric
ammonium citrate helps in the detection of H2S production.
(c) Sulphide-Indole-Motility Medium [SIM-Medium]. In fact,
the SIM-medium is employed
exclusively for the following three
different tests, namely :
(i) production
of sulphides,
(ii) formation
of indole i.e., a metabolite product duly obtained
from the subsequent utilization of tryptophan, and
(iii) causation
of ‘motility’.
Precisely,
the SIM-Medium is used for making
out the differentiation of the various enteric
organisms.
In a
broader perspective it is invariably necessary to screen and examine a host of pathological specimens, food, and pharmaceutical products (including
dosage forms) to ascertain the presence of
staphylococci ; besides, specific
organisms that are solely responsible for causing serious food
contamina-tion/poisoning as well as systemic infections. There are two media that are used extensively,
such as :
(a) Selective Media for Enterobacteria [or Enteric Bacteria]. [Greek. enterikos means per-taining to
intestine]. In general, all help in the degradation of sugars by means of the Embden-Meyerhof Pathway (or EMP-Cycle] which ultimately cause cleavage of ensuing
pyruvic acid to yield formic acid in
the formic-acid fermentations. It has been established beyond any reasonable
doubt that in the selective media for enterobacteria a
surface-active agent serves as the
‘main selector’, whereas in the specific
staphylococcal medium the various
selectors happen to be : sodium chloride [NaCl] and lithium chloride [LiCl].
Staphylococci are found to be tolerant against
a ‘salt’ concentration extending ~ 7.5% (w/v) e.g., Mannitol
salt, Baired-Parker (BP), and Vogel-Johnson (VI) media.
Salient Features. The other
vital and important ‘salient features’ with
respect to the various other ‘principles’ concerning the selective media for staphylococci are
as enumerated under :
(1) Use
of a selective C-source viz., mannitol or sodium pyruvate (soluble salt) along with a suitable ‘buffer’.
(2) Use
of an appropriate acid-base indicator
e.g., methyl-red phenolphthalein, for distinctly visualizing the ensuing
metabolic activity.
(3) By
observing the ‘inference growth’.
(4) Lecithin (a phospholipid) present in the
egg yolk forms a vital ingredient of
Baird-Parker medium seems to undergo hydrolysis strategically by the
ensuing staphylococcal (i.e., esterase) activ-ity* in
order that the prevailing microorganisms are adequately encircled by a cleared (i.e., transpar-ent) zone in the rest of the opaque medium.
Based on
advanced, meticulous researches carried out on the ‘molecular analysis’, pseudomonads
have been duly reclassified, and consequently several former Pseudomonas species reallocated to new genera,
for instance : Burkholderia,
Stenotrophomonas and others.
Importantly,
these media solely depend upon the relative resistance of pseudomonas to the
par-ticular quaternary ammonium
disinfectant cetrimide ;
whereas, in certain recipes the incorporation of nalidixic acid i.e., an antibiotic, affords a reasonable
resistance to the pseudomonads.
Laboratory Diagnosis. The
bacterium, Pseudomonads, usually
grows rapidly on a plethora of media
thereby rendering the identification of the pigmented strains of the organism from the clinical samples rather
easy. However, it has been duly observed that almost 1/10th of the isolates may
be nonpigmented.
Interestingly,
two cardinal functionalities do
confirm as well as ascertain the presence of the Pseudomonads, namely : (a) prompt oxidase reaction, and (b) arginine
hydrolysis. A typical exam ple of such a media is as given below :
Cetrimide Agar Media [CA-Media]. It is
usually employed to isolate the
Pseudomonads from either faeces
or other specimens having mixed flora.
Special Note. Because Ps. aeruginosa occurs as a most ‘frequent contaminant’, the actual isolation of the ensuing bacillus from a given sample must not always be accepted as a granted possible
proof of its critical etiological
involvement. Repeated isolation processes, therefore, may have to be
carried out so as to help towards the actual confirmation for the prevailing diagnosis.
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