Isolation and identification of

bacteria
C.Sworna kumari.,M.Sc.,M.Phil
 Isolation of Bacteria
• Isolation:
• The importance of this step is to isolate pure colonies of bacteria. The streak plate is a qualitative
isolation method; quadrant streaking is mostly done to obtain pure colonies.
The characteristics features of the colonies on solid agar media are then noted. This include

• Shape : circular, irregular or rhizoid.

• Size: small, medium, large( or in millimetres).
• Elevation: elevated, convex, concave, umbonate/umbilicate.
• Surface: Smooth, wavy, rough, granular, papillate or glistening.
• Edges: entire, undulate, crenated, fimbriate or curled.
• Colour: Yelow, green etc.( Note the colour of the colony).
• Structure: opaque, translucent or transparent.
• Degree of growth : scanty, moderate or profuse.
• Nature: discrete or confluent, filiform, spreading or rhizoid.
• In order to obtain the pure culture of organism, the isolated colonies are aseptically transferred on
to different nutrient agar slant tubes and incubated overnight at 37 degree Celsius. It is then stored
for future purpose.
 Isolation of individual cells from
mixed culture:

Materials Required:
• Mixed culture of organism.
• Inoculation loop
• Nutrient agar plate.
• Bunsen burner
• Incubator
• Procedure
• Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot.
• Take the test tube containing the nutrient broth with mixed culture of organism
that has been kept for 24 - 48 hours.
• Using aseptic technique loopful of organism from the broth.
• Quadrant streak the culture on the nutrient agar surface.
• Incubate the plates at 37 degree celcius for 24 hours.
• After incubation the isolated colonies are streaked aseptically on to nutrient agar
slant for further studies
 Simple Staining
• Staining is a simple basic technique that is used to
identify microorganisms. Simple staining is used
to study the morphology of all microorganisms.
• The simple stain uses the basic dyes such as
Methylene blue or basic fuschin.
• The strong negative charge of the bacterial cell
will strongly bind with the positive charged basic
dyes and will impart its colour to all
bacteria.
 Gram staining
• Gram staining is a differential staining technique that imparts different
colours to different bacteria or bacterial structures.
• Usually it differentiates bacteria into two groups; gram positive and gram
negative.
• The primary stain Crystal violet and mordent Iodine form a strong CVI
complex all bacteria. Gram positive cells due to their thick peptidoglycan
layer will retain the CVI complex even after it is subjected to
decolourization with acetone or alcohol. Hence the counter stain Safranin
has no action on gram positive cells.
• But in the case of gram negative, the thin peptidoglycan layer and more
lipid contents in the cell wall will easily make them susceptible to the
action of decolorizer and hence CVI complex is easily washed out and
hence the gram negative cells will the colour of counter stain
Safranin. Hence after the gram staining, the gram positive cells appear as
purple and gram negative cells appear as pink . The study of
morphological features and staining characteristics help in the preliminary
identification of the isolate.
 Biochemical reactions
• Biochemical reactions:
• Gram negative enteric bacilli play an important role in
the contamination of food.
• Hence they are the main causative agents of intestinal
infection. Gram negative family includes Shigella,
Salmonella, Proteus,
Klebsiella,Escherichia,Enterobacter etc.
• Usually four tests are used for differentiation of the
various members of Enterobactericeae. They are Indole
test,Methyl red test, Voges proskauer test and Citrate
test; collectively known as IMViC series of reactions.
 Indole test
• Indole tests looks for the presence or absence of
tryptophanase enzyme production of the
bacteria. If the enzyme is present, it will degrade
the aminoacid tryptophan in the media and will
produce Indole, ammonia and pyruvic acid.
• Indole will react with Kovac's reagent to produce
a cherry red complex, which indicates a positive
indole test. The absence of red color is indicative
of tryptophan hydrolysis due to the lack of
tryptophanse enzyme
 Methyl Red Test

• This test detects the ability of microorganism to ferment

glucose and to produce acidic end products. Enteric
organism produces pyruvic acid from glucose metabolism.
• Some enteric will then use the mixed acid pathway to
metabolize pyruvic acid to other acidic products such as
lactic acid, acetic acid and formic acids.
• This will reduce the pH of the media. Methyl red is a pH
indicator which is red at the acidic pH (below 4.4) and
yellow at alkaline pH (above 7).
• The formation of red color after the addition of Methyl red
reagent indicates the accumulation of acidic end products
in the medium and is an indicative of positive test
 Voges Proskauer Test
• This test determines the ability of microorganism to
ferment glucose. The end products of glucose
metabolism,pyruvic acid, is further metabolized by using
Butylene glucol pathway to produce neutral end such as
acetoin and 2,3 butanediol.
• When Barrit's reagent A ( 40% KOH) and Barrit's reagent B
(5% solution of alpha naphthol) is added it will detect the
presence of acetoin, the precursor in the 2,3- butanediol
synthesis.
• Acetoin in the presence of Oxygen and Barrit's reagent is
oxidized to diacetyl, where alpha naphthol act as a catalyst.
Diacetyl then reacts with guanidine components of
peptone to produce a cherry red colour
 Citrate Utilization Test

• This test determines the ability of microorganism to utilize Citrate.

• Some bacteria have the capability to convert the salts of organic
acids, for example, Sodium citrate to alkaline carbonates. Sodium
citrate is one of the important metabolite of Kreb's cycle.
• Certain bacteria use citrate as the sole carbon source. Citrate
utilization requires a specific membrane transporter and citrate
lyase activity. Citrate is converted to Oxalo acetic acid by citrate
lyase and oxaloacetate decarboxylase activity will convert
oxaloacetate to pyruvate with the release of carbondioxide.
• The other products of the reaction are acetate, Lactic acid, formic
acid etc. The carbondioxide reacts with sodium and water to form
sodium carbonate
 Triple sugar ion test
• The triple sugar- iron agar test is designed to differentiate among the
different groups or genera of the Enterobacteriaceae, which are all gram
negative bacilli capable of fermenting glucose with the production of acid,
and to distinguish them from other gram negative intestinal bacilli.
• This differentiation is based on the differences in carbohydrate
fermentation patterns and hydrogen sulfide production by the various
groups of intestinal organisms.
• Carbohydrate fermentation is detected by the presence of gas and a
visible colour change (from red to yellow) of the pH indicator, phenol red.
The production of hydrogen sulfide is indicated by the presence of a
precipitate that blackens the medium in the butt of the tube.
• TSI Agar contains three fermentative sugars, lactose and sucrose in 1%
concentrations and glucose in a concentration of 0.1%. Due to the building
of acid during fermentation, the pH falls. The acid base indicator Phenol
red is incorporated for detecting carbohydrate fermentation that is
indicated by the change in colour of the medium from orange red to
yellow in the presence of acids.
 TSI
• In case of oxidative decarboxylation of peptone, alkaline products are built and
the pH rises. This is indicated by the change in colour of the medium from orange
red to deep red.
• Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects
the production of hydrogen sulfide (indicated by blackening in the butt of the
tube).
• To facilitate the detection of organisms that only ferment glucose, the glucose
concentration is one-tenth the concentration of lactose or sucrose.
• The small amount of acid produced in the slant of the tube during glucose
fermentation oxidizes rapidly, causing the medium to remain orange red or revert
to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of
the tube since it is under lower oxygen tension.
• After depletion of the limited glucose, organisms able to do so will begin to utilize
the lactose or sucrose. To enhance the alkaline condition of the slant, free
exchange of air must be permitted by closing the tube cap loosely. If the tube is
tightly closed, an acid reaction (caused solely by glucose fermentation) will also
involve the slant.
 Urease test
•
• Urea is a nitrogen containing compound that is produced during the
decarboxylation process of the amino acid arginine in the urea
cycle. Urea is highly soluble in water and is thus it is an efficient way
for the human body to excess nitrogen.
• This excess urea is then taken away from the body with the help of
the kidneys as a part of urine. Certain bacteria produce the enzyme
urease during its metabolism process and that will break down the
urea in the medium to ammonia and carbon dioxide:
• Some enteric bacteria produce the enzyme urease, which splits the
urea molecule into carbon dioxide and ammonia. The urease test is
useful in identifying the genera Proteus, Providentia, and
Morganella, which liberate this enzyme.
 Urease test
• Urease, which is produced by some micro organisms, is an enzyme that is
especially helpful in the identification of Proteus vulgaris, although other
organisms may produce urease, their action on the substrate urea tends to
be slower than that seen with Proteus species. Therefore this test serves
to rapidly distinguish members of this genus from other lactose non
fermenting enteric micro organisms.
• Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond
in amide compounds such as urea and forms the alkaline end product
ammonia. The presence of urease is detectable when the organisms are
grown in a urea broth medium containing the pH indicator phenol red. As
the substrate urea is split into its products, the presence of ammonia
creates an alkaline environment that causes the phenol red to turn to
deep pink. This is a positive reaction for the presence of urease. Failure of
deep pink colour to develop is evidence of a negative reaction.
•
 Sulphide indole motility test
• The ability of an organism to move by itself is called motility. Motility is closely
linked with chemotaxis, the ability to orientate along certain chemical gradients.
Eucaryotic cells can move by means of different locomotor organelles such as cilia,
flagella, or pseudopods.
• Prokaryotes move by means of propeller-like flagella unique to bacteria or by
special fibrils that produce a gliding form of motility. Almost all spiral bacteria and
about half of the bacilli are motile, whereas essentially none of the cocci are
motile.
• The medium mainly used for this purpose is SIM medium ( Sulphide Indole
Motility medium) which is a combination differential medium that tests three
different parameters, sulphur reduction, indole production and motility. This
media has a very soft consistency that allows motile bacteria to migrate readily
through them causing cloudiness.
• In soft agar tubes non-motile bacteria will only grow on the inoculated region.
Motile bacteria will grow along the stab line and will tend to swim out away from
the stabbed area. Therefore, a negative result is represented by growth in a
distinct zone directly along the stab. A positive result is indicated by diffuse or
cloudy growth mainly at the top and bottom of the stabbed region.
 Gelatin Hydrolysis test
• Gelatin, a protein derived from the animal protein collagen.
• Some microorganisms possess an enzyme called gelatinase, which breaks
down gelatin into amino acids. Gelatin deeps contain the substrate gelatin,
which is a protein produced by the hydrolysis of collagen. Organisms
which hydrolyze gelatin will cause the gelatin to liquefy.
• The gelatin hydrolysis tests for an organism's ability to break down the
protein gelatin which is derived from collagen. Gelatin causes the media
to thicken, especially at cooler (below 28oC) temperatures.
• If the organism can release gelatinase enzymes the gelatin is broken down
or liquefied. The media is checked over a period of about a week after
inoculation and incubation at room temperature, for gelatinase
activity. The tube is placed on ice for a few minutes and if the media fails
to solidify it is considered a positive test. The gelatinase reaction may be
slow or incomplete
 Nitrate Reduction Broth
• Bacterial species may be classified into different groups depends on their
ability to reduce nitrate to nitrite or nitrogenous gases provided in the
growth medium.
• The reduction in nitrate can be coupled to anaerobic respiration in some
bacterial species. Nitrate, present in the broth, is reduced to nitrite
which is then reduced to nitric oxide, nitrous oxide, or nitrogen.
• The basis of nitrate reduction test the detection of nitrite and its ability to
form a red colored compound when it reacts with reagent A which
is sulfanilic acid and to form a complex (nitrite-sulfanilic acid) which then
reacts with Reagent B which is α-naphthylamine to give a red precipitate
(prontosil).
• Zinc powder act as a catalyst and that will favours the reduction of nitrate
to nitrite. Nitrate reaction occurs only under anaerobic conditions
• The medium is then transferred in tubes to make a low surface area to
depth ratio that will limit the diffusion of oxygen into the growth medium.
Most bacteria utilize the available oxygen in the medium for their growth
and will rapidly produce anaerobic conditions for the further reactions.
 Catalase Test
• The inability of strict anaerobes to synthesize catalase, peroxidase, or superoxide dismutase may
explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the
toxic concentration of H2O2 cannot be degraded when these organisms are cultivated in the
presence of oxygen. Organisms capable of producing catalase rapidly degrade hydrogen peroxide
which is a tetramer containing four polypeptide chains, which are usually 500 amino acids long. It
also contains four porphyrin heme groups(ie., iron groups) that will allow the enzyme to react with
the hydrogen peroxide.
• The enzyme catalase is present in most cytochrome containing aerobic and facultative anaerobic
bacteria. Catalase has one of the highest turnover numbers of all enzymes such that one molecule
of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen in a
second.
• Catalase production and activity can be detected by adding the substrate H2O2 to an appropriately
incubated (18- to 24-hour) tryptic soy agar slant culture. Organisms which produce the enzyme
break down the hydrogen peroxide, and the resulting O2 production produces bubbles in the
reagent drop, indicating a positive test. Organisms lacking the cytochrome system also lack the
catalase enzyme and are unable to break down hydrogen peroxide, into O2 and water and are
catalase negative.
 Coagulase Test
• Coagulases are enzymes that clot blood plasma by a mechanism
that is similar to normal clotting.
• The coagulase test identifies whether an organism produces this
exoenzyme. This enzyme clots the plasma component of blood. The
only significant disease-causing bacteria of humans that produce
coagulase are Staphylococcus aureus.
• Thus this enzyme is a good indicator of the pathogenic potential of
S. aureus. In the test, the sample is added to rabbit plasma and held
at 37° C for a specified period of time.
• Formation of clot within 4 hours is indicated as a positive result and
indicative of a virulent Staphylococcus aureus strain. The absence of
coagulation after 24 hours of incubation is a negative result,
indicative of an avirulent strain.
•
 Oxidase Test

Oxidase test is an important differential procedure that

should be performed on all gram-negative bacteria for their
rapid identification.
The test depends on the ability of certain bacteria to
produce indophenol blue from the oxidation of dimethyl-p-
phenylenediamine and α-naphthol.
This method uses N,N-dimethyl-p-phenylenediamine
oxalate in which all Staphylococci were oxidase negative. In
presence of the enzyme cytochrome oxidase (gram-
negative bacteria) the N,N-dimethyl-p-phenylenediamine
oxalate and α-naphthol react to indophenol
blue. Pseudomonas aeruginosa is an oxidase positive
organism.
 Starch Hydrolysis Test
• Amylases are a class of enzymes that are capable of digesting these glycosidic linkages found in starches. Amylases
can be derived from a variety of sources. Amylases are present in all living organisms, but the enzymes vary in
activity, specificity and requirements from species to species and even from tissue to tissue in the same organism.
Alpha-amylase (1,4 alpha D-Glucan-glucanohydrolase) acts upon large polymers of starch at internal bonds and
cleaves them to short glucose polymers.
• Alpha-amylase catalyzes the hydrolysis of internal Alpha-1-4 glucan bonds in polysaccharides containing 3 or more
alpha 1-4 linkages; it results in a mixture of maltose and glucose. Amyloglucosidase works on the shorter polymers
and splits off single glucose sugars. Bacterial alpha-amylase is particularly suited for industrial usage since it is
inexpensive and isothermally stable.
• Starch agar is an example of differential medium which tests the ability of an organism to produce certain alpha-
amylase and oligo-1, 6-glucosidase that hydrolyze starch. Starch molecules are too large to enter into the bacterial
cells, so some bacteria will secrete exoenzymes that will degrade starch into subunits that can be then easily
utilized by the organism.
• Starch agar is a simple nutritive medium with starch added. Since no colour change occurs in the medium when
organisms hydrolyze starch, iodine solution is added to the plate after incubation. Iodine turns blue, purple, or
black (the colour depends on the concentration of the iodine used) in the presence of starch. A clearing around
the bacterial growth shows that the organism has hydrolyzed starch.
• Lipid Hydrolysis:
• Trybutyrene agar is used for the detection and enumeration of lipolytic microorganisms in food and other
material