Selective and Diagnostic Media

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Chapter: Pharmaceutical Microbiology : Identification of Microorganisms

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.



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 :


1. Differential Media


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 :


Eosin Methylene Blue Agar [EMB-Agar]


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.


MacConkey Agar


It has already been discussed under Section 3.


Hektoen Enteric Agar [HE-Agar]


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.


2. Enrichment Media


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 :


Blood Agar*


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.


Chocolate Agar


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.


3. Characteristic Media


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 :


Triple Sugar Iron Agar [TSI Agar]


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.


A. Selective Media for Staphylococci


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.


B. Selective Media for Pseudomonads


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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