Extensive and meticulous in depth investigations carried out on a host of fermentative procedures
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 belowwith appropriate explanations whenever required in the course of the prevalent discussion :
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
.
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 disruption
by virtue of the gas evolved (usually termed as
‘stormy fermentation’).
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 acidtryptophan 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.
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 represents
a
negative test.
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/
v) 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.
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 pertains
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.
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.
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.
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.
Production of Hydrogen Sulphide (H
2S)
Importantly, there are several S-containing amino acids
e.g., cystine, cysteine, methionine that
may decompose certain organisms to yield H
2S (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 :
Pb(CH
3CO)2 + H2S ⎯→ PbS↓ + 2CH3COOH
Lead Acetate Hydrogen Lead Acetic Acid
sulphide sulphide
(Black)
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. 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.
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 negative)
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.
Oxidase Reaction
The underlying principle of the
‘oxidase reaction’ is exclusively by virtue of an enzyme knownas 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 applicator
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.
Egg-Yolk Reaction
It has been duly demonstrated and proved that all such organisms which essentially and specifically
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.
Growth in Presence of Potassium Cyanide (KCN)
Occasionally, buffered liquid-culture medium containing KCN in a final concentration of approximately
1/13,000 (
i.e., 7.69 × 10–5) is employed critically to identify certain KCN-tolerant enteric
bacilli
.
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 enumerated
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 areduly 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 predominantly
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 sometimes
to carry out the identification of certain critical organism(s). Now, with the advent of ever-increasing
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 examined
; 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 andmethodical diagnosis of organism from the patients specimen.