1. Identification of unknown bacteria is an important task for microbiologists that involves determining what taxon the bacteria belongs to. 2. There are three main categories of identification methods - phenotypic/morphological analysis, immunological/serological techniques, and genetic/molecular methods. 3. Staining techniques like simple staining, gram staining, and acid-fast staining are used for morphological analysis and involve using dyes to distinguish cell structure and characteristics. Biochemical tests like IMViC are also used to identify bacteria based on enzymatic reactions and metabolite production.

1. IDENTIFICATION
OF
BACTERIA

2. INTRODUCTION
• Identification is the process of determining to which established
taxon a new isolate or unknown strain belongs.
• Identification of unknown bacterial culture is one of the major
responsibilities of a microbiologist
• Samples of blood, tissue, water, food and cosmetics are examined
daily in laboratories throughout the world for the presence of
contaminants and pathogenic microorganisms.
• Pharma industries and research institutes are constantly screening soil,
water, marine samaples to isolate new antibiotics, enzymes and
vitamins producing microorganisms.
• Bergey’s Manual of Systemic Bacteriology (BMSB) has been the
official, internationally accepted reference for bacterial classification.
• In the current edition of Bergey’s manual, bacteria is classified into 33
groups called sections and contain information of 4000 bacterial
species.
• Link: https://www.goshen.edu/bio/Biol206/Biol206Labs/ident.pdf

3. Why Identification is important?
• Medical diagnostics & clinical significance
— identifying a pathogen isolated from a
patient.
• Food & brewage industries — identifying a
microbial contaminant responsible for food
spoilage and fermentation.
• Research setting — identifying a new
isolate which carries out an important
process.

4. IDENTIFICATION METHODS
• The methods fall into three
categories:
Phenotypic/Morphology (micro
and macroscopic)
Immunological/Serological
analysis
Genetic techniques/Molecular
method

5. STAINING REACTIONS
 To study size, shape, arrangement and
properties and different specific groups of
microorganisms, Biological stains are used.
Stains is an organic compounds containing a
benzine rings with chromophore and
auxochrome group.
Different types of staining techniques are used
to study the morphological and structural
properties of microorganisms.

6. STAINING TECHNIQUES
1. SIMPLE STAINING
2. NEGATIVE STAINING
3. GRAM STAINING
4. ACID-FAST STAINING
 BIOLOGICAL TESTS(IMViC Test)

7. Understanding with examples

8. Dyes
 Basic dyes: Methylene blue, Basic fuchsin, Crystal violet,
Safranine, Malachite green.
• Have positively charged groups (usually from penta-valent
nitrogen).
• Generally sold as chloride salts.
• Basic dyes bind to negatively charged molecules such as nucleic
acids, many proteins and surfaces of bacterial and archeal cells.
• Used for Positive staining
 Acidic dyes: Eosin, Rose Bengal, Nigrosin(indian ink), Congo red
and Acid fuchsin possess groups such as carboxyls (-COOH) and
phenolic hydroxyls (-OH).
• Acidic dyes, in their ionized form, have a negative charge and bind
to positively charged cell structures.
• Used for negative staining

9. 1. SIMPLE STAINING/Monochrome Stains
• Used only single stain
• Ex: Any one at a time- Methylene blue(2-3mins), Crystal violet(1-
2mins), Carbol fuchsin(15-30secs), Safranin(1mins), Malachit
green(1-2mins), etc.
• Used to study Size, shape and bacterial cell arrangements.
• Basic stains with a positively charged chromogens (any substance
that can become a pigment or coloring matter, as a substance in
organic fluids that forms coloured compounds when oxidized, or a
compound, not itself a dye, that can become a dye) are used.
 Purpose: To elucidate the morphology and arrangement of
bacterial cells.

10. Steps involved in simple staining

11. PROCEDURE
Select oil/grease free slide. Do it by washing with
detergent and wiping the excess water and then dry the
slide by passed through flame.
These slide is allowed to air dry and smear of sample is
applied.
After air drying these slide is rapidly passed through a
flame for three to four times for heat fixation.
After heat fixation the slide is flooded with a particular
stain and these stain is allowed to react for two-three
minutes.
Further the slide is washed under running distilled water.
The slide is air dried and watched under oil immersion
microscope.

12. Mechanism behind the simple staining
1. A stain has a ability to bind a cellular component .These
abilities depend upon the charges present on cellular
component and charges present on chromophore group
of stain.
2. Bacteria has large number of carboxyl group on its
surface and these carboxyl group has negative charge.
3. When these carboxyl group carry out ionization reaction it
shows COO– and H+
COOH Ionisation COO– + H+
4. In nature, these H+ ions (unstable) are present on cell
surface and further replaced by other positively charged
ions like Na+ or k+ and H+ bonds with oxygen to form
water.

13. • Thus, surface of an unstained bacterial cell is represented as
• Basic dyes are available as salts of acids
Eg: Methylene blue chloride(MB.Cl, Malachite green
chloride(Mg.Cl)
When these are ionised,
MB.Cl Ionisation MB+ + Cl-
• On addition to methylene blue for staining, exchange of MB+
with Na+ on the bacterial cell take place.
• Thus, when colouring agent forms ionic bond with cell or cell
components, it results into the staining of cell.
• Concentration of MB is 0.5-2% in water.

14. NEGATIVE STAINING
• Colouring the Background of Object
• Not stain but made visible against dark background
• Bacteria are mixed with acidic stains such as Eosin
or Nigrosin that provide a uniformly coloured
background against which the unstained bacteria
stand out in contrast.
• Useful to observed bacteria that are difficult to stain
(Spirili & spirochetes– Trepanoma palladium,
Borrelia burgdorferi, Leptospira) and in
demonstration of bacterial capsule.
• Acidic stains has negative charge; therefore, it
doesn’t combine with negatively charged of bacteria
cell surface.
Advantages over Simple:
1. Natural size and shape of microorganism can be
seen
2. Heat fixation is not required
3. Doesn't need physical and chemical treatment.
4. It is possible to observe bacteria that are difficult
to stain.

16. HERO OF GRAM STAINING
Dr. HANS CHRISTIAN GRAM:
• Danish bacteriologist noted for his development of the Gram stain
in 1884.
• It is used to differentiate bacterial species into two broad groupes ,
Gram positive & Gram negative based on the physical property of
their cell wall.

17. GRAM STANING
• It is not only reveals the size and shape of
bacteria but also used to differentiate bacteria
into Gram positive and Gram negative cells.
Hence, called differential staining.
• It is first and usually the only method employed
for the diagnostic identification of bacteria in
clinical specimens.
• Provides more information about the
characteristics of the cell wall (Thickness).
• A stain is a chemical substance that adheres to a
cell, giving the cell colour.

18. Why heat fixation?
• To preserve the shape of
the cells or tissue
• To prevent autolysis of
the cell
• To adhere the bacterial
cell on the slide properly
• To kill unwanted
microbe attached to the
edge of the slide.

19. Principle
• Violet dye and the iodine combine to form an insoluble, dark
purple compound in the bacterial protoplasm and cell wall.
• This compound is dissociable in the decolourizer, which
dissolves and removes its two components from the cell.
• But the removal is much slower from Gram-positive than from
the Gram-negative bacteria, so that by correct timing the former
stay dark purple whilst the latter become colourless.
• The difference between the two types of bacteria is that the
Gram positive have thicker and denser peptidoglycan layers in
their cell walls, which makes them less permeable to the stain
than those of the Gram negative bacteria.
• The iodine has a critical role in enhancing this difference.
• It seems to bind temporarily to the peptidoglycan and make it
even less permeable to the dye.

20. Structure of Gram positive and Gram
negative bacteria

22. Procedure
• Preparation of smear
• Air dry and Heat fixation

24. Mechanism behind the Gram staining
1. Primary stain(Crystal violet):
 Crystal violet stain is used first and stain all cells deep violet in
colour.
 Concentration: 0.1 %
2. Mordant(Gram’s iodine): Not a stain
 Any substance that forms an insoluble compound with stain and serve
to fix the colour to bacterial cell.
 It lead to form insoluble complex- Crystal Violet Iodine-Magnesium
ribonucleate [CV-I-Mg ribonucleate] complex in gram-positive
bacteria.
 This complex is not formed in Gram negative bacteria, as Mg-
ribonucleate is absent in the cell wall. Due to this, CV-I complex is
formed in Gram negative bacteria.
 Preparation: Dissolve 20 g of Potassium iodide in 250 ml water & then
add 10 g iodine. When iodine is dissolved, make upto 1 ltr with water.

25. 3. Decolourising agent (ethyl alcohol, 95%, acetone,
analine):
Gram positive cell contains more Mucopeptide
(Peptidoglycan)/ Lipoteichoic acid (LTA)
On application of decolourising agents Like alcohol,
acetone, aniline, etc, shrinkage of cell wall take
place due to dehydration and decreases the
permeability for CV-I-Mg ribonucleate complex
(insoluble).
Thus, the complex is retained in the cell and hence
cell is stained deep violet in colours.
In Gram negative, there is increase in permeability
property of porin present in cell wall. Due to this,
CV-I complex is extracted and cell gets decolourised
(lose violet colour)

26. 4. Counter stain (Safranin):
• Safranin is used as counter stain in Gram staining
procedure to differentiate between gram positive
and gram negative organisms.
• They are crystalline solids showing a
characteristic green metallic lustre;
• Basic red 2
• Colouring all cell nuclei red.
• 0.5% in distilled water.
• Link: http://himedialabs.com/TD/S027.pdf

27. Examples

29. HERO OF ACID FAST STAINING
• Dr. PAUL ERHLICH, was a German-Jewish physician
• He is credited with finding a cure for syphilis
• In 1908, he received the Nobel Prize in Physiology or
Medicine for his contributions to immunology.9.

30. ACID FAST STAINING
• Another widely used differential staining procedure in bacteriology
developed by Ehrlich in 1882.
• Also known as Ziehl–Neelsen staining.
• Some bacteria(specially M. Tuberculosis, Nocardia asteroides,
Actinomycetes, and M. leprae) resist toward the gram staining or other
staining due to high lipid content in the cell wall, hence they are called
ACID FAST BACTERIA.
• Because of high lipid content, acid fast cell have low permeability to
dyes and hence difficult to stain.
• 5% aquouse phenol and heat(physical intensifier) are used to
enhance the penetration of primary stain(chemical intensifier)
• Classified into two group-
 A) ACID FAST BACTERIA
 B) NON ACID FAST BACTERIA
• Acid-fast cells contain a large amount of lipids and waxes in their cell
walls
• Primarily mycolic acid

31. PROCEDURE
 A clean sterile glass slide was taken.
 A thin uniform smear was prepared in the slide using a inoculation loop.
 The smear was allowed to air dry and then heat fixed.
 The smear was flooded with Ziehl-Neelsen Carbol Fuchsin (ZNCF) and
heated until steam arises.
 Preparation was allowed to stay for 5-7 min. The stain must not be
allowed to evaporate or dry in the slide. Pour more carbol fuchsin on
slide is necessary.
 It is then washed under a low steam of running water.
 The smear was decolorized with 20% sulphuric acid OR 3% HCl + 95%
ethanol.
 The slide is then washed again under running distilled water.
 Counter stain the smear with methylene blue/Malachite green for the 2
min.
 Washed under running tap water.
 Slide was blot dried and examined under microscope.

37. • SCHAEFFER AND FULTON METHOD:
• Spore staining, Malachit green (primary stain) and
counter stain(safranin).
• Albert’s stain:
• Link:https://microbeonline.com/albert-stain-principle-
procedure-results-uses/
• https://www.microrao.com/alberts_staining.htm
• https://microbeonline.com/albert-stain-principle-
procedure-results-uses/

38. Biochemical Test for bacteria
IMViC and TSI
• IMViC:
• Indole: Break down the amino acid Tryptophan
• Methyl Red: Glucose oxidation
• Voges-Proskauer: Production of neutral end products
• Citrate: Citrate fermentation
• Some microorganism are differentiate on the basis of
enzyme-catalysed metabolic reactions
• Presence and absence of certain enzymes, intermediately
metabolites end products often give valuable
information in identifying and classifying microorganism.
• These tests are useful in distinguishing members
of Enterobacteriaceae(Aerobic, non-acid fast, gram
negative bacilli found in human and other animal intestine.

39. Indol Production test
• Identifies bacteria capable of producing indole
• Some bacteria are capable of converting tryptophan (an amino acid) to
indole and pyruvic acid by using the enzyme tryptophanase
• Pyruvic acid can be converted to energy or used to synthesize other compounds
required by the cell
Principle Reaction:

40. Procedure
• Indol productionis detected by
inoculating the test microorganisms
into peptone water and incubating it
at 37°C for 2-5days.
• Then add 0.5ml of Kovac’s
reagent(10drops) and mix gently.
• If indole is produced, A “cherry-red”,
band forms on the surface of the
media.
• Motility (if present) can be seen as
growth of the bacteria away from the
stab line
• Sulfur in the media may be reduced to
hydrogen sulfide (H2S); this appears as
a “blackening” within the media

41. Interpretation:
• Development of cherry red colour at the
interface of the reagent and the broth, within
seconds after adding the Kovacs’ reagent
indicates the presence of indole and the test is
positive.
• If no colour change is observed, then the test is
negative and so organisms are not capable of
producing indol.

42. Methyl red (MR) Test
• Used to detect the production of acid during fermentation of
glucose.
• Mainly used to differentiate between E.coli and E. aerogen
• By production of acid, pH of the medium falls and maintained
below 4.5
• Methyl red is a pH indicator (red at pH less than 4.4 and yellow at a
pH greater than 6).
• MR-VP broth tubes(2-3No.)
Principle Reactions:

43. Procedure
• Inoculate the test microorganisms in glucose
phosphate broth and incubate at 37°C for 2-
5days.
• MR-VP medium- (Glucose phosphate peptone
water)
• Then add five drops of 0.04% solution of
methyl red, mix and observe the result in the
form of change in colour.

44. Interpretation
• A red colour indicates that
glucose has been oxidized.
• Methyl red positive tube on
the right(Acid production)-
E.coli
• Methyl red negative tube on
the left(No acid production)-
E. aerogen

45. Voges-Proskauer Test
• Used to determine the ability of microbes to produce non-acidic or neutral
end products (acetyl methyl corbinol- acetoin, 2,3-butanediol and Diacetyl).
• MR-VP broth is a combined medium used for two tests—Methyl Red and
Voges-Proskauer.
• This test is characterizes E.aerogen
• MR-VP broth tubes
• In the alkali medium and oxygen, the amount of acetyl methyl carbinol present
in the medium is oxidised to diacetyl which react with the peptone of the broth
to give a red colour.
Principle Reactions:

46. Procedure
• Inoculate test microorganisms in glucose
phosphate broth (two test tubes) and
incubate at 37°C for 48hrs.
• MR-VP medium- (Glucose phosphate
peptone water)
• Then add 1ml of 40% potassium hydroxide
and 3ml of 5% solution of α-naphthol in
absolute alcohol. Barritt’s Reagent (10drops
in each test tube)-shake
• Stand for 5-10mins for colour development

47. Interpretation
• Development of crimson
red colour indicates
positive test for
E.aerogen.
• And no colour change
indicates negative test.

48. Citrate Utilization Test
• Used to determine if an organism is capable of fermenting
citrate as the sole source of carbon for growth.
• It indicated by the production of turbidity in the medium.
• The ability of an organism to utilize citrate occurs via the
enzyme citrase.
Principle Reaction:

49. Procedure
• Reagent: Bromothymol
blue
• Media: Simmon’s Citrate
Agar
Result:
• +ve: Blue
• -ve : Green

51. Write short note on the following:
• Nitrate reduction test
• Hydrogen sulphide production
• Potassium cynide test
• Catalase production
• Urease test
• Oxidase test
• Sugar fermentation
• Litmus milk reactions

52. Triple sugar iron agar(TSI)
This test is used to determine the ability to:
1- Reduce sulfur into H2S.
2- Lactose & /or Sucrose fermentation.
3- Glucose fermentation and Gas production.
 Used to differentiate among the different groups of
Enterobacteriaceae
Media Used: Triple sugar-iron agar
• TSI medium contains:
• 1% lactose , 1% sucrose & 0.1% glucose.
• Sodium thiosulfate & ferrous sulfate (iron).
• Phenol red as indicator (to detect acid production from
carbohydrate fermentation ).

53. Procedure:
• Inoculate TSI
medium with an
organism by
inoculating needle
by stabbing the
butt & streaking
the slant, incubate
at 37°C for 24
hours.

54. Reading Results:

56. • Red slant/Red butt = No fermentation
• Red slant/Yellow butt = Only glucose fermentation
• Yellow slant/yellow butt = Lactose and/or sucrose
fermentation
• Dark color: Hydrogen Sulfide produced Sodium
thiosulfate reduced