BIOCHEMICAL TESTS FOR BACTERIA

Prado, R., Tarun, N.A.

Department of Biology
College of Science
University of the Philippines Baguio

ABSTRACT
The morphological and biochemical method of identification of bacteria is the classical method
of characterization of bacteria. Due to the characteristics of the bacteria such as cell shape, spore
forming capabilities, whether they are aerobic or anaerobic or Gram positive or Gram negative,
they are conveniently grouped into a number assemblages. Diversely, bacteria’s morphological
attributes are not enough constituent for them to be identified, thus, biochemical tests and other
methodologies are developed. In this experiment, four isolates have been tested with the different
biochemical tests. Media are prepared for the different tests and the four isolates are inoculated
to each medium. Then, after the incubation period needed every test, the reaction of the bacteria
to each medium is observed. Hence, the isolates showed positive result to some tests after
executing the biochemical tests. However, isolates ensued a negative result to most of the tests
being executed. With this essence, identification of the bacteria to genus or species level can be
done.

INTRODUCTION
Bacteria, archaea, fungi, algae, protozoan and protists are integral part of microbial

diversity and these seem to be an unnoticed normal resource that deserves greater attention.

Microbial diversity is the variety that exists among microorganisms and their environments.

Microorganisms are found in all ecosystems. The bacteria are remarkable in the abilities to live

in environments that are hospitable for life and the greatest among energy sources. (Jarvis &

Goodacre, 2008)

Bacteria may be conveniently grouped into a number of natural assemblages based

characteristics such as cell shape, spore forming capabilities and whether they are

aerobic/anaerobic or Gram positive / Gram negative (Manero & Blanch, 1999). The

morphological and biochemical method of identification of bacteria is the classical method of

characterization of bacteria. Classical identification of individual bacterial species in

environmental samples typically involves isolation, laboratory culture and then taxonomic

characterization. The classification of bacteria into families, genera and species is based on a

wide range of phenotypic characteristics (Baird-Parker, 1963). These include culture conditions,

colony morphology, biochemical characteristics and detailed morphology.

Moreover, during decades, bacteria have been routinely identified in laboratories by

biochemical tests namely catalase test, oxidase test, casein hydrolysis, gelatine hydrolysis,

hydrogen sulphide production, indole test, and starch hydrolysis and by microscopic

identification, which, as a rule, need time for their execution, ranging from a couple of hours to

several days. Conventional analyses are time-consuming techniques and semi-automatic methods

require great amounts of biologic material (Tan, Ellis, Lee, Stamper, Zhang & Carroll, 2012).

Since bacteria do not have enough morphological characteristics to pinpoint their peculiarities,

several methodologies have been developed to test their metabolic or enzymatic reactions,

enabling grouping and identification at genus or species level (Alsina & Blanch, 1994).

Definitely bacteria produce certain types of metabolites when they grow on various types

of media thus standard biochemical tests are familiarized and used to characterize and identify

unknown bacteria.

MATERIALS AND METHODS

There are seven tests used during the experiment. In catalase test, a colony of the purified

culture was put in a sterilized slide and a drop of hydrogen peroxide is added in the colony.

While in oxidase test, a piece of whatman filter paper was dampen with a drop of 1% TPDS,

using a sterilized glass rod, a colony of bacteria is acquired and rubbed against the wet filter

paper. Into a sterile Petri plate, double strength Nutrient agar (NA) is prepared and mixed with
one tube of skim milk for casein hydrolysis. After the agar had set and dried, the plate is divided

into four parts and in each quadrant, four purified culture of bacteria is inoculated and incubated

for 42 hours at 37°C. In a prepared tube of nutrient gelatine, purified culture of bacteria is stab

inoculated and incubated at 37°C for two days. For an hour, it was refrigerated.

Slants of triple sugar iron (TSI) are prepared for the testing of hydrogen sulphide

production. Into the surface of the slant, the bacteria are introduced using fishtail inoculation.

Then, the butt of the slant medium is stabbed up to 1⁄4 inch from the bottom using an inoculating

needle. It was then incubated for 24 hours at 37°C. For indole test, tubes of 0.1% tryptone broth

are prepared. And, it was inoculated with the bacteria and incubated for a day at 37°C. After the

incubation period, one mL of xylene is added and was shaken until the xylene arose. Then, 0.5

mL of Erlich’s reagent is added. Lastly, a plate of pre-poured basal medium with soluble starch

is prepared. The plate was divided into four quadrants and in each quadrant four different

purified cultures are introduced and inoculated. Then, it was incubated for 24 hours at room

temperature. After incubating, the plate is flooded with Lugol’s iodine and clearing zone had

been observed.

RESULTS AND DISCUSSION

Fundamental scientific studies in microbiology and similar disciplines utilize correctly

identified bacterial species to draw reliable inferences and conclusions in researches. Bacterial

identification is a complex task, which is why several methods were developed for easier

identification of such microorganisms, including biochemical tests. These tests are inexpensive,

reliable, easily applied in all laboratories and include catalase test, oxidase test, casein, gelatin,

and starch hydrolysis, hydrogen sulfide production, and indole test, among others (Alaee et. al,

2015).
Catalase Test

The oxidoreductase enzyme catalase plays a crucial role in quenching reactive oxygen

species such as hydrogen peroxide often produced as a by-product of aerobic respiration. The

enzyme is found in a wide range of aerobic and anaerobic organisms, acting as an antioxidant

and protecting the bacterial cells against oxidative stress (Arya et. al, 2018).

Such enzyme is fundamental in catalase test for bacterial species and especially useful in

identifying gram positive cocci (Duben-Engelkirk & Engelkirk, 2008). It is essential in

differentiating between bacterial genera, the speciation of some gram positive and gram negative

bacteria, and in distinguishing between aerobic and anaerobic bacetria which generally lack the

enzyme (Reiner, 2010). Catalase test involves adding a drop of hydrogen peroxide to a bacterial

sample on a slide and observing for the formation of bubbles, which indicates positive reaction.

Bacterial isolates tested for catalase both produced bubbles after addition of hydrogen

peroxide, signifying the presence of the enzyme in the bacterial cells. From such results, it can be

inferred that the isolates are gram positive cocci and can be classified as either aerobic or

facultative anaerobic species, although further tests to confirm such inference is required.

Fig. 1. From the left, catalase test results for bacterial isolates A and B.

Hydrogen peroxide is a highly reactive and damaging molecule which accumulates

whenever oxygen participates in various metabolic reactions. Catalase neutralizes the effects of

the chemical and effectively removes hydrogen peroxide by catalyzing the breakdown of the
molecules into water and oxygen, manifested in the formation of bubbles (Duben-Engelkirk &

Engelkirk, 2008).

The basic mechanism in the reaction of catalase with hydrogen peroxide involves the

breakdown and subsequent breakdown of the reactive oxygen species of H2O2 into oxygen and

water following the reaction below (Arya et. al, 2018).

2 H2O2 Catalase 2H2O + O2

(Substrate)(Enzyme) (Products)

As a dismutase enzyme, catalase contains a heme moiety at the active site and converts

the two hydrogen peroxide molecules into oxygen and water. The small amount of hydrogen

peroxide binds at the active site to generate catalase compound I, which then reacts with a

second molecule of hydrogen peroxide, releasing oxygen and water (Kehrer et. al, 2010).

Oxidase Test

Oxidase is an abbreviated name for cytochrome oxidase, an enzyme which participates in

the electron transport chain (Duben-Engelkirk & Engelkirk, 2008). Cytochrome c oxidases are

the terminal enzymes in the respiratory chains of mitochondria and many bacterial species,

whose electrons reduce molecular oxygen to produce water (Behr et. al, 1998). Such enzyme is

used in oxidase test, which reacts with tetramethyl-phenylenediamene-dihydrochloride (TPDS)

to produce a purplsih blue coloration.

Oxidase test is useful for initial categorization of many bacterial species composed of

distinct colonial morphology (Koneman, 2006).The oxidase test detects the presence of a

cytochrome oxidase system which catalyzes the transport of electrons between electron donors in

the bacteria and a redox dye tetramethyl-p-phenylene-diamine, reducing the dye into a deep
purple color. The cytochrome system is usually present only in aerobic organisms capable of

utilizing oxygen as the final electron acceptorsuch as Pseudomonas, Neisseria, Alcaligens,

Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella (Aryal, 2018).

The absence of purplish-blue coloration signifies negative results for the test, manifested

by the bacterial isolates tested in the laboratory. Application of TPDS reagent in a filter paper

rubbed with the bacterial species showed no color change thus interpreted as a negative result.

Oxidase negative isolates thuscan be inferred as facultative anaerobic, as the bacterial species

produced positive results with catalase test but negative in oxidase test, which is used to identify

strict aerobic organisms. Essentially, negative results in the test imply that the isolates do not

belong to aforementioned bacterial genera, thus belonging to another taxon.

Fig. 2. From the left, isolates A and B with oxidase-negative results

Intracellular oxidase enzyme present in cytochrome containing organisms catalyzes the

oxidation of cytochrome c, and the TPDS reagent added acts as an artificial electron acceptor for

the enzyme oxidase (Aryal, 2018). The purple color is caused by indophenol, formed from the

oxidation of the oxidase reagent (Duben-Engelkirk & Engelkirk, 2008).

Casein Hydrolysis

Casein is the most important component found in milk containing a rich source of

different essential amino acids. Casein is composed of three polypeptide chains held together by

non-covalent interactions and is responsible for the white, opaque appearance of milk (Kalyankar

et. al, 2016). Such protein is used in casein hydrolysis test where bacterial species are allowed to

grow in a media composed of nutrient agar and skim milk as source of casein and a postive

results is indicated by the appearance of clearing zones.

The protein casein is way too large to enter the cell membrane, thus must undergo step by

step degredation into peptones, polypeptides, dipeptides, and amino acids before assimilation

inside the cell. Such process is mediated by extracellular enzymes proteases, which cleave the

peptide bond CO-NH by introducing water, liberatiing the low molecular weight amino acids to

be transported inside the cell (Harrigan, 1998). Organisms secreting proteases exhibits a zone of

proteolysis once introduced in a media with casein. Such zone is represented by a clear area

surrounding the bacterial growth, which is a result of a hydrolytic reaction yielding souble, non-

colloidal amino acids.

Laboratory results of tested bacterial isolates showed negative reaction to casein

hydrolysis marked by the absence of clearing zones. Instead, it can be carefully observed that the

bacterial species flooded the medium with growth. Such indicates that the isolates are negative

for casein hydrolysis, thus lack the enzyme proteases which degrade the protein.

Gelatin hydrolysis

The gelatin hydrolysis test detects the ability of bacteria to produce gelatinases, proteases

secreted extracellularly to hydrolyze or digest gelatin. Gelatin hydrolysis aids in the

identification of Serratia, Pseudomonas, Flavobacterium, and Clostridium, distinguishes

gelatinase-positive Staphylococcus aureus from gelatinase-negative Staphylococcus epidermidis,

and between gram postive, spore-forming, rod shaped, aerobic or anaerobic bacteria such

asBacillus anthracis, B. cereus, B. subtilis, Clostridium perfringens and C. tetani (dela Cruz &

Torres, 2012).

Gelatin is derived from collagen, a connective tissue among vertebrates and is produced

when collagen is boiled in water. Gelatin is used to create a nutrient gelatin medium, which

detects gelatin hydrolysis and serves as both the solidifying agent and substrate for gelatinase

activity. Upon stab-inoculationof gelatinase positive bacteria, secreted gelatinases hydrolyze the

gelatin by degrading it into polypeptides and fruther converting into amino acids, resulting in the

liquefaction of the medium. Refrigeration of the medium is required to obtain accurate results,

where gelatinase positive result remains in liquid form, while negative in gelatin hydrolysis

solidifies (dela Cruz & Torres, 2012).

Experimental results after execution of helatin hydrolysis test reveal that bacterial isolates

are gelatinase negative, manifested in the lack of liquefaction in the gelatin medium. A solidified

medium is more evident instead, indicating the the isolates lack gelatinases which digests the

gelatin medium.

Fig. 3. From the left, isolates A and B those were negative in gelatin hydolysis
Hydrogen sulfide production

The capacity to produce hydrogen sulfide has been widely used as a diagnostic test for

bacteria (Kuster &Williams, 1963). Microorganisms are able to metabollize sulfur and release

hydrogen sulfide under aerobic conditions (Hou et. al, 2017).Such ability to reduce sulfur

containing compounds during metabolism is employed to test microorganisms for identification.

Testing for hydrogen sulfide gas production includes an iron and sulfur compound in the

test medium, which produces hydrogen sulfide once sulfur is reduced. Hydrogen sulfide

production is performed by certain bacteria through reduction of sulphur containing amino acids

like cystine, methionine or through the reduction of inorganic sulphur compounds such as

thiosulfates, sulfates or sulfites during protein degradation or when anaerobic respiration shuttles

the electrons to sulfur instead of to oxygen. In either case, hydrogen sulfide gas is produced

which reacts with the iron compound to form the black precipitate of ferric sulfide. The black

color acts as an indicator for the presence of hydrogen sulfide (Hou et. al, 2017).

Oftentimes, hydrogen sulfide production test employs the use of triple sugar iron agar,

which differentiates organisms on the basis of dextrose, sucrose, and lactose fermentation and

hydrogen sulfide production. Color changes in the butt and slant portion of the medium indicate

different results due to the reaction of the bacteria in eaach of the components present in the agar.

Hydrogen sulfide production test of bacterial isolates show that samples A and B

manifested different colors, thus vary in the type of carbohdrate fermented. Isolate A appears

yellow and red on the slant and butt part respectively, while isolate B exhibites a red butt and

yellowish orange to almost red slant.

 Fig. 4. Isolates A and B results in hydrofen sulfide production

The differences in color are due to the fermentation of carbohydrate detected by the

production of gas and visible color change of the pH indicator, phenol red. Thus, a yellow, acidic

butt and red, alkaline slant indicates fermentation of dextrose. A yellow color in both the slant

and the butt implies fermentation of dextrose, lactose, and/or sucrose, while a red slant and butt

indicate that the bacteria is a non-fermenter. Gas production observed in the media is manifested

by the splitting and cracking of the agar while hydrogen sulfide production results in a black

precipitate at the bottom of the tube. While isolate A contains an acidic slant which implies

fermentation of dextrose, lactose or sucrose, the alkaline butt is a misleading result, thus further

test is required. Meanwhile, isolate B with yellowish-orange to almost red slant and butt imply

that the bacterial isolate introduced is a non-fermenter. Both bacterial species tested lack the

formation of a black precipitate at the bottom, thus negative in hydrogen sulfide production.

Indole test

The indole test screens the ability of bacteria to degrade the amino acid tryptophan to

produce indole. The test was first used in 1889 to distinguish between Escherichia coli and

Enterobacter aerogenes, until variations were made in the test to characterize coliforms

(MacWilliams, 2009). Currently, the test is important in the identification of Enterobacteria.

 Tryptophan is an amino acid that undergoes hydrolysis by bacteria containing

tryptopahanase, splitting the molecule into indole following the reaction presented below.Indole

is generated by reductive deamination from tryptophan via the intermediate molecule

indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine

group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic

acid, ammonium and energy. Pyridoxal phosphate is required as a coenzyme.

Fundamental in the conduct of indole test is the Ehrlich reagent, a more sensitive

compound used as an alternative to Kovac’s reagent. Ehrlich reagent is recommended when

testing bacterial groups that produce little indole such as nonfermentativebacilli or anaerobes.

The reagent is added after placing xylene in the tryptophan broth to create a purplish-red ring in

the xylene layer to indicate positive reaction.

Indole test for both bacterial isolates in the laboratory garnered negative results, hence,

no formation of a purplish-red ring is observed. This implies that the isolates do not contain

trptophanase responsible for converting the tryptophan amino acid into indole.
 Fig. 5. From the left, isolates A and B with indole-negative result

Starch Hydrolysis

Starch is a complex polysaccharide found abundantly in plants and usually deposited in

the form of large granules in the cytoplasm of the cell. Starch consists of 2 components—

amylose and amylopectin, which are present in various amounts. The amylose consists of D-

glucose units linked in a linear fashion by α-1,4 linkages. It has 2 non-reducing ends and a

reducing end. Amylopectin is a branched polysaccharide. In these molecules, shorter chains of

glucose units linked by α-1,4 are also joined to each other by α-1,6 linkages. The major

component of starch can be hydrolyzed by a-amylase, which is present in some bacteria while

well known in case of fungi. The ability to degrade starch is used as a criterion for the

determination of amylase production by a microbe (Tille, 2014).

In addition, many bacteria produce extracellular enzymes used to catalyze chemical

reactions outside of the cell. In this manner, nutrient sources, such as starch, that are too large to

be absorbed through the cell membrane can be broken down into smaller molecules and

transported into the cell via diffusion (Acharya, 2013).

 In the starch hydrolysis test, the test bacteria are grown on agar plates containing starch.

If the bacteria have the ability to hydrolyze starch, it does so in the medium, particularly in the

areas surrounding their growth while the rest of the area of the plate still contain non-hydrolysed

starch. Since no color change occurs in the medium when organisms hydrolyze starch, iodine

solution is added as an indicator to the plate after incubation. While the non-hydrolysed starch

forms dark blue color with iodine, its hydrolyzed end products do not acquire such dark blue

color with iodine. Consequently, transparent clear zones are formed around the colonies that

hydrolyze starch while the rest of the plate show a dark blue coloration as iodine forms the

colored complex with starch (Aryal, 2019).

Out of the four isolates, only one showed a clear zone after flooding the plate with

Lugols’s iodine. Thus, the bacteria have the capability to produce amylase.

Fig. 6. Starch hydrolysis test result of four different isolates

SUMMARY AND CONCLUSION

Bacteria do not have enough morphological characteristics to pinpoint their peculiarities.

For this reason, several tests such as catalase test, oxidase test, casein hydrolysis test, gelatin

hydrolysis test, hydrogen sulfide production test, indole test, and starch hydrolysis have been

developed in order to assess their metabolic or enzymatic reaction that one may identify and
group these microorganisms at genus or species level. Different tests have been executed in four

different isolates after the preparation of the media needed and after transferring those isolates to

the respective media in each test. Then, negative results and positive results have been observed

and examined. The results indicate that different species of bacteria have different reaction

towards the tests being accomplished. It can also be concluded that through these tests, the genus

or species of these isolates can be identified.

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