Biochemical Tests (or Properties)

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Chapter: Pharmaceutical Microbiology : Identification of Microorganisms

The most vital and important and abundantly employed biochemical tests are as described below with appropriate explanations whenever required in the course of the prevalent discussion :



Extensive and meticulous in depth investigations carried out on a host of fermentative proce-dures using different types of substrates exclusively dependent upon a broad-spectrum of biochemical tests ultimately lead to the production of ethanol by yeast ; acetylmethylcarbinol ; lactic acid ; acetic acid ; ethanol by E. coli ; acetone plus CO2 ; citric acid (Krebs Cycle) ; and CO2 plus H2.


The most vital and important and abundantly employed biochemical tests are as described below with appropriate explanations whenever required in the course of the prevalent discussion :


1. Carbohydrate (Sugar) Fermentation


The carbohydrate fermentation is normally tested in a ‘sugar media’. Thus, the generation of ‘acid’ is indicated by an apparent change in the colouration of the ensuing medium either to pink or red, and the resulting gaseous products produced gets duly collected in a strategically placed Durham’s Tube.


2. Litmus Milk


In this particular instance there may not be any change in the medium, or acid or alkali could be generated thereby giving rise to clotting of milk, and peptonizaiton or saponification may take place appreciably. The resulting ‘clot’ i.e., coagulation of the milk protein (viz., casein) could face a disrup-tion by virtue of the gas evolved (usually termed as ‘stormy fermentation’).


3. Indole Production


In actual practice the ‘indole production’ is normally tested in a peptone-water culture after an interval of 48 or 96 hours incubation at 37°C ; whereby the generation of indole from the amino acid tryptophan is duly ascertained as given below :

When Kovak’s Reagent*, 0.5 mL, is added carefully and shaken gently for a while, it yields a red colouration thereby indicating a positive reaction i.e., indole production.


4. Methyl Red Test [MR-Test]


The MR-test is frequently used to carry out the detection for the ‘production of acid’ in the course of fermentation of glucose, besides maintaining pH below 4.5 in an old culture medium [methyl red : 4.2 (red) to 6.3 yellow].


Procedure : Five droplets of methyl red solution [0.04% (w/v)] are added into the culture in glucose-phosphate medium that had been previously incubated at 30°C for 5 days, mixed well, and read instantly. Appearance of red colour (acidic) gives a positive test, whereas yellow colour repre-sents a negative test.


5. Voges-Proskauer Test [VP-Test]


The underlying principle of the VP-Test exclusively rests upon the production of acetyl methyl carbinol from pyruvic acid via an intermediate stage in its strategic conversion to form 2, 3-butylene glycol i.e., [CH3CH-(OH)CH(OH)CH3]. However, it has been duly observed that in the presence of alkali and atmospheric oxygen (O2) the relatively small quantum of acetyl methyl carbinol present in the medium gets oxidized to the corresponding ‘diacetyl derivative’ that subsequently interacts with the peptone content in the ‘culture broth’ to produce a distinct red colouration.


Procedure : The VP-Test may be easily performed by the careful addition of 0.6 mL of a 5% (w/


solution of α-naphthol in ethanol and 0.2 mL solution of 40% (w/v) KOH to 1 mL of a glucose phosphate medium culture of the ensuing organism previously incubated duly either at 30°C for a duration of 5 days or at 37°C for 2 days. Thus :


Positive Reaction : indicated by the appearance of a pink colouration in just 2-5 minutes, that ultimately gets deepened either to magenta or crimson red in about 30 minutes duration ;


Negative Reaction : Designated by the appearance of a colourless solution upto 30 minutes.

Importantly, the development of any traces of pink colouration must be ignored completely.


6. Citrate Utilization


In actual practice, Koser’s citrate medium containing ‘citric acid’ serves as the exclusive source of carbon. Evidently, the ability as well as the efficacy for the ‘citrate utilization’ (i.e. , the prevailing substrate) is adequately indicated by the production of reasonably measurable turbidity in the medium.


Note : The various biochemical characteristic tests viz., indole, MR, VP, and citrate are quite useful in the proper and prompt identification of Gram-negative microorganisms. Hence, these tests are frequently referred to by the Sigla ‘IMVIC’ Tests.


Alternatively, another cardinal physiological difference that may be exploited specifically per-tains to the ensuing ‘growth temperature’. It has been duly demonstrated that at 44°C only A. aerogenes shall display growth particularly, whereas E. coli will not. Therefore, the specific incubation at 44°C shall be able to make a clear cut distinction between these two microorganisms which is invariably known as the Eijkman (E) test. The menomic i.e., aiding the memory is IMVEC, wherein E stands for Eijkman. Conclusively, the four cardinal tests are normally distinguished by mnemonic IMVIC or when the Eijkman test is also included, IMVEC, and several texts predominantly refer to the IMVIC or IMVEC characteristic features of these and other, related organisms.


Summararily, therefore, the apparent behaviour of the said two microorganisms may be stated as below, whereby a sort of comparison between E. coli and A. aerogenes has been recorded :


7. Nitrate Reduction


The ‘nitrate reduction’ test is carried out after allowing the specific bacterium to grow for 5 days at 37° C in a culture broth containing potassium nitrate [1% (w/v)]. The test reagent comprises of a mixture of equal volumes of the solutions of sulphanilic acid and α-naphthylamine in acetic acid carefully mixed just before use. Now, 0.1 mL of the test reagent is duly incorporated to the culture medium. The results of the test may be inferred as given under :


Positive Reaction : Development of a red colouration within a short span of a few minutes confirms a positive reaction.


Negative Reaction : The critical absence of the above mentioned red colouration signifies a negative reaction.


Importance : The ‘nitrate reduction’ test indicates particularly the presence of the enzyme nitrate reductase that helps to reduce nitrate to nitrite.


8. Ammonia Production


The ‘ammonia production’ test is usually performed by incorporating very carefully the Nessler’s Reagent* into a peptone-water culture grown meticulously for 5 days at 37°C. The inferences of this test may be drawn as stated under :


Positive Test : Appearance of a Brown colour ;


Negative Test : Appearance of faint Yellow colour.


9. Urease Test


The ‘test’ is usually carried out in Christensen’s Urea-Agar medium or Christensen’s urease medium.


Procedure. The slope is inoculated profusely and incubated at 37°C. The slope is duly examined at intervals of 4 hours and 24 hours incubation. The test must not be taken as negative till after a duration of 4 days after incubation.


Result : The urease positive cultures give rise to a distinct purple-pink colouration*. The exact mechanism may be explained by virtue of the fact that urease producing microorganisms largely help in the conversion of urea to ammonia** (gas) which is particularly responsible for the desired colouration.


10. Production of Hydrogen Sulphide (H2S)


Importantly, there are several S-containing amino acids e.g., cystine, cysteine, methionine that may decompose certain organisms to yield H2S (gas) amongst the products of microbial degradation. In this particular instance lead acetate [Pb(CH3CO)2]*** is duly incorporated into the culture media which eventually gets turned into either black or brown due to the formation of PbS as given below :


Procedure : The organisms are grown in culture tubes. In actual practice a filter-paper strip soaked in a lead acetate solution [10% (w/v) freshly prepared] is strategically inserted between the cotton plug and the empty-space in the culture tube.


Result : The gradual browning of the filter paper strip rightly confirms the H2S-production.


11. Reduction of Methylene Blue


The reduction of 1 drop of the aqueous methylene blue reagent [1% (w/v) added into the broth culture and incubated at 37°C. The results are as indicated below :


Strongly positive : exhibited by complete decolourization


Weakly positive : displayed by green colouration.


12. Production of Catalase [Tube catalase Test]


In this specific test a loopful (either a wooden applicator stick or a nichrome wire loop) H2O2 i.e., hydrogen peroxide (3%) is placed meticulously right upon the colonies grown on the nutrient agar medium. The catalase production is indicated by the prompt effervescence of oxygen (O2) due to the fact that the enzyme catalase aids in the conversion of H2O2 into water and oxygen bubbles (in the form of effervescence).


Importance : It has the unique means of differentiation between Streptococcus (catalase nega-tive) from Staphylococcus (catalase positive).


Caution : Such ‘culture media’ that specifically contain blood as an integral component are definitely not suitable for the ‘tube catalase test’ because the blood itself contains the enzyme catalase.


13. Oxidase Reaction


The underlying principle of the ‘oxidase reaction’ is exclusively by virtue of an enzyme known as cytochrome oxidase that particularly catalyzes oxidation of reduced cytochrome by oxygen.


Procedure : A solution of tetramethyl p-phenylene diamine dihydrochloride [concentration 1.0 to 1.5% (w/v)] is poured gently as well as carefully over the colonies. The result is duly indicated by the oxidase positive colonies turning into maroon-purple-black in a span of 10 to 30 minutes.


Kovac’s Method : Alternatively, the ‘oxidase reaction’ may also be performed by Kovac’s method. In this method a strip of filter paper is adequately moistened with a few drops of 1% (w/v) solution of tetramethyl p-phenylene diamine dihydrochloride. By the help of a sterilized wooden appli-cator the actual growth from an agar medium is carefully smeared onto the exposed surface of the said strip of filter paper. Thus, a positive test is invariably indicated by the distinct development of a purple colouration almost promptly (within 10 seconds).


Importance : The obvious importance of the ‘oxidase reaction’ is judiciously employed to obtain a clear cut differentiation/separation of the enterics from the pseudomonads.


Example : Pseudomonads aeruginosa : Positive Test.


Escherichia coli : Negative Test.


14. Egg-Yolk Reaction


It has been duly demonstrated and proved that all such organisms which essentially and specifi-cally produce the enzyme lecithinase e.g., Clostridium perfringens, on being carefully grown on a solid egg-yolk medium, gives rise to well-defined colonies usually surrounded by a zone of clearing.


15. Growth in Presence of Potassium Cyanide (KCN)


Occasionally, buffered liquid-culture medium containing KCN in a final concentration of ap-proximately 1/13,000 (i.e., 7.69 × 10–5) is employed critically to identify certain KCN-tolerant enteric bacilli.


16. Composite Media


In the domain of ‘Biochemical Tests’ the pivotal role of composite media is gaining legitimate recognition for the particular identification of biological isolates.


Advantages : The various cardinal advantages of the so called composite media are as enumer-ated under :

·        it serves as an economical and convenient culture media ; and

·        a ‘single composite medium’ strategically indicates different characteristic properties of the bacterium (under investigation) that otherwise necessarily might have required the essential usage of several individual cultural media.


Examples : The two most commonly employed ‘composite media’ are as described under :


(a) Triple Sugar Iron Medium (TSI-Medium) : It represents a rather popular ‘composite medium’ that specifically indicates whether a bacterium under investigation :

·        ferments glucose exclusively,

·        ferments either, lactose or sucrose,

·        gas formation occurs or not, and

·        indicates production of H2S gas.

In actual practice, TSI-medium is distributed in various tubes along with a butt and a slant. After having subjected them to proper innoculation under perfect asceptic conditions one may draw the following inferences :


Red slant + Yellow butt. indicates that all sugars viz., glucose, lactose, and sucrose are duly fermented.


Appearance of bubbles in the butt—shows production of gas, and


Blackening of the medium—displays evolution of H2S gas in the TSI-Agar Reaction.


Importance : The most spectacular and major advantages of the TSI-medium is to predomi-nantly facilitate the preliminary identification of the Gram-negative Bacilli.


(b) Test for Amino Acid Decarboxylation : The specific biochemical test essentially involves the ‘decarboxylases’ (viz., arginine, lysine, ornithine) ; and the phenomenon of decarboxylation of the amino acids invariably gives rise to the corresponding release of amine and CO2. In reality, this particular test is solely employed for the identification of enteric bacteria.


In conclusion, one may add that there are certain other tests as well, namely ; fermentation of organic acids, hydrolysis of sodium hippurate, and oxidation of gluconate which are used some-times to carry out the identification of certain critical organism(s). Now, with the advent of ever-increas-ing wisdom and knowledge pertaining to the plethora of metabolic processes in the growth of various microorganisms, the number of reliable tests also is increasing progressively.


Note : One may consult the ‘special referred manuals’ to have an access to the detailed descriptions as well as actual utilities of these tests.


Biochemical Tests for Identification of Bacterial Isolates : After having accomplished the microscopic and the critical growth characteristic features of a pure culture of organisms are duly exam-ined ; highly precise and specific ‘biochemical tests’ may be carried out to identify them exactly. Based on the survey of literature and genuine evidences from various researches carried out one may come across certain ‘biochemical tests’ usually employed by most clinical microbiologists in the proper and methodical diagnosis of organism from the patients specimen.


A few such typical examples are summarised duly in the following Table : 4.1.


Table : 4.1. Specific Biochemical Tests Carried out by Clinical Microbiologists for the Critical Diagnosis of Microorganisms Derived from the Patient’s Specimen Directly

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